Cell proliferation reducing cancer specific PCNA peptides

ABSTRACT

Cell-permeable caPCNA-derived peptides and their variants serve as therapeutic compositions to reduce the proliferation of cancerous cells and also augment cytotoxic effects of chemotherapeutics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims priority toInternational Application Serial No. PCT/US2009/051643 filed Jul. 24,2009, which claims priority to a U.S. Provisional Application Ser. No.61/083,393 filed Jul. 24, 2008, the entire disclosures of which areincorporated herein by reference. This application additionally claimspriority under 35 USC §119(e) to U.S. Provisional Application Ser. No.61/249,528 filed on Oct. 7, 2009, the entire disclosure of which isincorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made with government support under CA012189 awardedby the National Institutes of Health and W81XWH-07-1-0707 awarded by theDepartment of Defense. The U.S. government has certain rights in theinvention.

TECHNICAL FIELD

This disclosure relates to peptide-based therapeutic compositions andmethods to inhibit cancer cell proliferation.

BACKGROUND

Proliferating cell nuclear antigen (PCNA) plays an important role in theprocess of DNA replication, repair, chromosomal recombination, cellcycle check-point control and other cellular proliferative activities.In conjunction with an adaptor protein, replication factor C (RFC), PCNAforms a moving clamp that is the docking point for DNA polymerases deltaand epsilon. Different isoforms of proliferating cell nuclear antigen(PCNA) that display both acidic and basic isoelectric points (pI) havebeen demonstrated. Analysis of PCNA by two-dimensional polyacrylamidegel electrophoresis (2D PAGE) from both malignant and non-malignantbreast cells (referred to as non-malignant PCNA or nmPCNA) and tissuesrevealed the presence of an acidic form of PCNA only in malignant cells(referred to as the cancer-specific PCNA or csPCNA or caPCNA). Thisdifference in isoelectric points between these two forms of PCNA,appears to result from an alteration in the ability of the malignantcells, to post-translationally modify the PCNA polypeptide and is notdue to a genetic change within the PCNA gene.

Structural work examining the structure of the PCNA polypeptide todefine the structural differences between the caPCNA and non-malignantcell isoform of PCNA revealed a region of the caPCNA protein that isuniquely exposed only in the cancer cell. An antibody was developed to aregion of the cancer specific isoform of PCNA that is highly selectivefor the PCNA isoform expressed exclusively in cancer cells.

Proliferating cell nuclear antigen (PCNA) is a 29 kDa nuclear proteinand its expression in cells during the S and G2 phases of the cellcycle, makes the protein a good cell proliferation marker. It has alsobeen shown to partner in many of the molecular pathways responsible forthe life and death of the cell. Its periodic appearance in S phasenuclei suggested an involvement in DNA replication. PCNA was lateridentified as a DNA polymerase accessory factor in mammalian cells andan essential factor for SV40 DNA replication in vitro. In addition tofunctioning as a DNA sliding clamp protein and a DNA polymeraseaccessory factor in mammalian cells, PCNA interacts with a number ofother proteins involved in transcription, cell cycle checkpoints,chromatin remodeling, recombination, apoptosis, and other forms of DNArepair. Besides being diverse in action, PCNA's many binding partnersare linked by their contributions to the precise inheritance of cellularfunctions by each new generation of cells. PCNA may act as a mastermolecule that coordinates chromosome processing.

PCNA is also known to interact with other factors like FEN-1, DNAligase, and DNA methyl transferase. Additionally, PCNA was also shown tobe an essential player in multiple DNA repair pathways. Interactionswith proteins like the mismatch recognition protein, Msh2, and thenucleotide excision repair endonuclease, XPG, have implicated PCNA inprocesses distinct from DNA synthesis. Interactions with multiplepartners generally rely on mechanisms that enable PCNA to selectivelyinteract in an ordered and energetically favorable way.

The use of short synthetic peptides for the generation of polyclonal andmonoclonal antibodies has been successful. Peptides are known to serveas chemo-attractants, potent neurological and respiratory toxins, andhormones. The peptides have also been used as affinity targets andprobes for biochemical studies, and have provided a basis forunderstanding the characteristics and specific nature of discreteprotein-protein interactions. In addition, peptide hormones exert potentphysiological effects, and in some cases the active hormone is either apeptide that is contained within a larger protein or is processed andreleased from a precursor protein prior to exerting its physiologicaleffect.

Peptides have been used to disrupt protein-protein interactions, byacting as highly specific competitors of these interactions. Biochemicalstudies employing peptide reagents advanced the use of peptides astherapeutic drugs capable of disrupting cell functions that requireprotein-protein interactions. Thus, specific cellular processes such asapoptosis and cell cycle progression, which are dependent upon discreteprotein-protein interactions, can be inhibited if these protein-proteininteractions are selectively disrupted. The replication of genomic DNAbeing dependent on protein-protein interactions is also susceptible topeptide-induced inhibition of these protein interactions.

In vivo DNA synthesis is a highly regulated process that depends on amyriad of biochemical reactions mediated by a complex series ofprotein-protein interactions. Cell division is dependent on the DNAsynthetic process, and cancer cell growth is substantially sensitive toany agent that disrupts the regulation and/or the activity of the DNAsynthetic machinery responsible for copying the cancer cell's genomicDNA. In addition, it was demonstrated that one signature of breastcancer is the induction of genomic instability, as transformed cellsdevelop a highly aggressive metastatic phenotype. Genomic instabilityarises through a series of changes in the cellular DNA syntheticmachinery that alters the fidelity with which DNA is synthesized.

Studies utilizing the carboxyl terminal 26 amino acids from the p21cipprotein, (which is known to interact with the PCNA protein),demonstrated the ability of this peptide to disrupt the cellularproliferative process. This peptide fragment of p21 potentially disruptsone or more cellular processes utilizing PCNA and presumably interfereswith critical protein-protein interactions that participate in the DNAsynthetic process as well as the regulation of other cell cyclecheck-point controls and the induction of apoptosis.

Studies utilizing this peptide fragment of p21 have demonstrated theability of the p21 peptide to activate a non-caspase associatedapoptotic pathway. Similarly, studies involving a 39 amino acid peptidefragment of the p21 protein partially inhibited DNA replication in vivo,and suggest that this peptide fragment of p21 can stabilize the PCNA-p21protein interaction leading to the decrease in DNA synthetic activitywithin the cell.

In addition, computational chemical methods are being used to modelspecific regions of the PCNA molecule that may interact with othercellular proteins involved in cell cycle check point control and DNAsynthesis. Regions of the cyclin-CDK complex may serve as templates toidentify target sites for disrupting key cell cycle check-point controlpoints that are essential for cell proliferation.

Use of synthetic peptides to inhibit cell proliferation and the processof selectively targeting cancer specific PCNA protein to mediate theinhibition of cell proliferation is needed to treat cancer.Peptidomimetic drugs that interact with an antigenic site or target siteon caPCNA to disrupt specific protein-caPCNA interactions that areunique to the cancer cell are desired. Peptides derived from caPCNAspecific epitopes, described herein, significantly augment the cytotoxiceffects of specific traditional chemotherapeutic regimens andconsequently kill cancer cells in a highly selective manner.

Germ-line mutations in BRCA1 or BRCA2 alleles are associated with a highrisk of the development of a number of cancers, including breast,ovarian, and prostate cancer. Cells lacking these or other important DNArepair proteins have deficiencies in the repair of DNA double strandedbreaks by homologous recombination. For example, loss of BRCA1 functionoften leads to aggressive tumors, and the tumors are often resistant tochemotherapeutic DNA damaging agents. Thus, novel therapeutics, such ascaPCNA peptides, that exploit the DNA repair defects in cancersharboring mutations in homologous recombination pathways, areadvantageous to standard chemotherapeutics used alone.

SUMMARY

A method of inducing cell death in a breast cancer cell or apre-malignant cell includes administering a therapeutically effectiveamount of a composition comprising a caPCNA peptide, wherein the caPCNApeptide comprises an amino acid sequence selected from the groupconsisting of LGIPEQEY (SEQ ID NO: 1), LAIPEQEY (SEQ ID NO: 2), LGIAEQEY(SEQ ID NO: 3), LGIPAQEY (SEQ ID NO: 4), LGIPEAEY (SEQ ID NO: 5),LGIPEQAY (SEQ ID NO: 6), LGIAEAEY (SEQ ID NO: 7), LGIPEAAY (SEQ ID NO:8), LGIAEQAY (SEQ ID NO: 9), and LGIAEAAY (SEQ ID NO: 10).

In an aspect, the cell harbors one or more mutations in a DNA repairprotein. In an aspect, the DNA repair protein participates in homologousrecombination. Illustrative examples of homologous recombination repairproteins include, but are not limited to BRCA1, BRCA2, or PALB2, RAD51,RAD52, XRCC3, MRE11, and the like. Illustratively, the cancer cell orpre-malignant cell may be a breast, ovarian, or prostate cell.

A method of reducing cellular proliferation of a breast cancer cell or apre-malignant cell of an individual includes administering atherapeutically effective amount of a composition comprising a caPCNApeptide capable of disrupting caPCNA interaction with one or more of itsbinding partners and wherein the individual has one or more BRCA1mutations.

Peptide variants derived from specific regions or domains of cancerspecific (caPCNA)-interacting proteins interfere with the interaction ofcellular proteins with the PCNA protein in vivo and thereby affectcancer cell survival. Specific amino acid sequences representing peptidefragments of the caPCNA protein disrupt the regulatory activity of PCNAand subsequently inhibit cancer cell growth through the disruption offunctioning of cellular processes that require PCNA, including DNAreplication, repair, chromosomal recombination, and cell cyclecheck-point control.

Amino acid substitutions in one or more positions improve the cytotoxiceffects of caPCNA-derived peptides. caPCNA-derived peptides includingamino acid substituted peptides that have tags or domains that enhancecellular uptake, increase the cytotoxic effects of the caPCNA peptides.

A therapeutic composition for reducing cellular proliferation ofmalignant cells includes a peptide molecule having an amino acidsequence selected from the group consisting of LGIPEQEY (SEQ ID NO: 1),LAIPEQEY (SEQ ID NO: 2), LGIAEQEY (SEQ ID NO: 3), LGIPAQEY (SEQ ID NO:4), LGIPEAEY (SEQ ID NO: 5), LGIPEQAY (SEQ ID NO: 6), LGIAEAEY (SEQ IDNO: 7), LGIPEAAY (SEQ ID NO: 8), LGIAEQAY (SEQ ID NO: 9), and LGIAEAAY(SEQ ID NO: 10). In an embodiment, the peptide molecule is a syntheticmolecule.

A therapeutic composition for reducing cellular proliferation ofmalignant cells includes a peptide molecule consisting essentially of anamino acid sequence selected from the group consisting of LGIPEQEY (SEQID NO: 1), LAIPEQEY (SEQ ID NO: 2), LGIAEQEY (SEQ ID NO: 3), LGIPAQEY(SEQ ID NO: 4), LGIPEAEY (SEQ ID NO: 5), LGIPEQAY (SEQ ID NO: 6),LGIAEAEY (SEQ ID NO: 7), LGIPEAAY (SEQ ID NO: 8), LGIAEQAY (SEQ ID NO:9), and LGIAEAAY (SEQ ID NO: 10).

In an embodiment, the caPCNA-derived peptide molecules further include acell permeable factor or a cell-uptake agent. For example, the cellpermeable factor is a cell penetrating peptide selected from the groupof amino acid sequences RRRRRRR (SEQ ID NO: 11), RRRRRRRR (SEQ ID NO:12), RRRRRRRRR (SEQ ID NO: 13), RRRRRRRRRR (SEQ ID NO: 14), RRRRRRRRRRR(SEQ ID NO: 15), RQIKIWFQNRRMKWKK (SEQ ID NO: 16), GRKKRRQRRRPPQ (SEQ IDNO: 17), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18), GRKKRRQRRR (SEQ IDNO: 19) or a factor listed on Table 4.

In some aspects, the cell penetrating peptide includes one or moreD-amino acids. Illustratively, the cell penetrating peptide may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more D-Arg residues.

In some aspects, the cell penetrating peptide is covalently linked orconjugated to the peptide molecules derived from caPCNA. In otheraspects, the cell penetrating peptide is recombinantly fused with thepeptide molecule. In certain embodiments, the cell penetrating peptidefurther includes a spacer sequence. The spacer sequence may be about1-10 or about 1-20 amino acids in length. Synthetic spacers are alsosuitable as long as they do not interfere with the translocation of thepeptide across the cell membrane.

It is appreciated herein that the therapeutic compositions may containone or more caPCNA derived peptides and/or variants, nuclearlocalization sequences, cell penetrating peptides, spacers, or anycombination thereof.

In an embodiment, the peptide is engineered to be protease resistantcompared to an unmodified native caPCNA-derived peptide. In anembodiment, the peptides disclosed herein are generated as retro-inversoisomers.

In an embodiment, the composition that includes a caPCNA-derivedtranslocatable peptide may further include a chemotherapeutic agent.Preferably, the chemotherapeutic agent is a DNA damaging agent.Illustrative examples of DNA damaging agents include, but are notlimited to, doxorubicin, irinotecan, cyclophosphamide, chlorambucil,melphalan, methotrexate, cytarabine, fludarabine, 6-mercaptopurine,5-fluorouracil, capecytabine, cisplatin, carboplatin, oxaliplatin,and/or combinations thereof.

It is appreciated herein that chemotherapeutic agent may be administeredto a cancer patient prior to, along with, or after the administration ofthe composition that includes the caPCNA-derived peptides or variantsthereof. It is also appreciated that one or more chemotherapeutic agentsmay be formulated together with one or more caPCNA peptides.

In an embodiment, the compositions that include the peptide inhibitorsof caPCNA interaction may be delivered as a liposome. The liposome mayalso include nanoparticles. In an aspect, one or more constituents ofthe pharmaceutical composition that includes caPCNA-derived peptides mayfurther include a nanoparticle.

A method to inhibit the growth of a cancer cell, the method includes:

-   -   (a) obtaining a therapeutically effective amount of the        composition that includes a peptide disclosed herein;    -   (b) administering the composition such that the agent contacts a        population of cancer cells; and    -   (c) inhibiting the growth of the cancer cell with the        composition.

In an embodiment, the composition is administered intravenously. Anymode of administration, including direct delivery to cancer is possible.A chemotherapeutic agent may be administered to a cancer patient priorto or along with or after the administration of the composition thatincludes the caPCNA-derived peptides or variants thereof. In anembodiment, radiotherapy may also be administered prior to or along withor after the administration of the composition that includes thecaPCNA-derived peptides or variants thereof. Radio therapy includes beamradiation therapy and radio isotope therapy.

A method of sensitizing cancer cells for cancer therapy includes:

-   -   (a) administering a therapeutically effective amount of the        composition that includes a peptide molecule comprising an amino        acid sequence selected from the group of LGIPEQEY (SEQ ID NO:        1), LAIPEQEY (SEQ ID NO: 2), LGIAEQEY (SEQ ID NO: 3), LGIPAQEY        (SEQ ID NO: 4), LGIPEAEY (SEQ ID NO: 5), LGIPEQAY (SEQ ID NO:        6), LGIAEAEY (SEQ ID NO: 7), LGIPEAAY (SEQ ID NO: 8), LGIAEQAY        (SEQ ID NO: 9), and LGIAEAAY (SEQ ID NO: 10), such that the        composition contacts a population of cancer cells to sensitize        the cancer cells; and    -   (b) administering a chemotherapeutic agent or radiotherapy to        inhibit the growth of the cancer cells.

A method of chemoprevention of cancer includes:

-   -   (a) administering a chemopreventative agent comprising the        composition of composition that includes a peptide molecule        comprising an amino acid sequence selected from the group of        LGIPEQEY (SEQ ID NO: 1), LAIPEQEY (SEQ ID NO: 2), LGIAEQEY (SEQ        ID NO: 3), LGIPAQEY (SEQ ID NO: 4), LGIPEAEY (SEQ ID NO: 5),        LGIPEQAY (SEQ ID NO: 6), LGIAEAEY (SEQ ID NO: 7), LGIPEAAY (SEQ        ID NO: 8), LGIAEQAY (SEQ ID NO: 9), and LGIAEAAY (SEQ ID NO:        10), such that the composition contacts a population of cells        that are likely to express cancer specific isoform of PCNA        (caPCNA); and    -   (b) preventing the transformation of the cells that express the        caPCNA isoform to a malignant state or to develop tumors.

In an embodiment, a chemopreventative agent is administered to anindividual considered high-risk for a cancer type. In an embodiment, thechemopreventative agent is administered to an individual withpre-cancerous cells.

Rational drug design methodologies can also be implemented to obtainspecific inhibitors of caPCNA cellular interaction based on thestructural or sequence information of a caPCNA derived peptide, e.g., apeptide that has an amino acid sequence LGIPEQEY (SEQ ID NO: 1). In anembodiment, the agent is a peptide fragment derived from anintracellular protein. In an embodiment, the intracellular protein isknown to interact with caPCNA.

A therapeutic composition for reducing in vivo cellular proliferation ofmalignant cells that express a cancer specific isoform of proliferatingcell nuclear antigen (caPCNA), the composition includes a peptidemolecule that has an amino acid sequence LGIPEQEY (SEQ ID NO: 1) with acell-permeable peptide sequence comprising a polyarginine sequence. Inan embodiment, the peptide domain that facilitates peptide uptake acrosscells is R9 (SEQ ID NO: 13) (nonarginine tag).

A liposome composition for reducing in vivo cellular proliferation ofmalignant cells that express a cancer specific isoform of proliferatingcell nuclear antigen (caPCNA), the composition includes a peptidemolecule comprising an amino acid sequence R9-LGIPEQEY (SEQ ID NO: 20)with one or more amino acid substitutions or a functionally equivalentstructure thereof or a peptidomimetic thereof, wherein R9 is eitherconjugated chemically or is part of a recombinant fusion protein.

A suitable cell surface targeting factor that is used along with one ormore of the compositions disclosed herein is selected from the group ofHER2/neu, estrogen receptor, progesterone receptor, epidermal growthfactor receptor (EGFR).

A recombinant cell that expresses a caPCNA-derived peptide, wherein thepeptide selectively disrupts protein-protein interaction in cancercells. In an embodiment, the caPCNA-derived peptide includes an aminoacid sequence LGIPEQEY (SEQ ID NO: 1) with one or more amino acidsubstitutions.

A synthetic peptide that includes an amino acid sequence LGIPEQEY (SEQID NO: 1) with one or more amino acid substitutions and a peptidetranslocation sequence.

Other suitable PCNA-derived peptide inhibitors wherein one or more aminoacids are substituted include QLGIPEQEYSC (SEQ ID NO: 21), VEQLGIPEQEY(SEQ ID NO: 22), LGIPEQEYSCVVK (SEQ ID NO: 23), LGIPEQEYSCVVKMPSG (SEQID NO: 24), EQLGIPEQEY (SEQ ID NO: 25), QLGIPEQEY (SEQ ID NO: 26),LGIPEQEYSCVVKMPS (SEQ ID NO: 27), LGIPEQEYSCVVKMP (SEQ ID NO: 28),LGIPEQEYSCVVKM (SEQ ID NO: 29), LGIPEQEYSCVV (SEQ ID NO: 30),LGIPEQEYSCV (SEQ ID NO: 31), LGIPEQEYSC (SEQ ID NO: 32), QLGIPEQEYSC(SEQ ID NO: 33), LGIPEQEYS (SEQ ID NO: 34) that have one or more aminoacid substitutions and combinations of the additional NH₂ and COOHtermini amino acids that flank LGIPEQEY (SEQ ID NO: 1) with one or moreamino acid substitutions and a cell-permeable sequence such as R9 (SEQID NO: 13).

Replication defective viral expression vectors (e.g., lentivirus,adenovirus, adeno-associated virus, herpes virus, and others) capable ofexpressing the peptides disclosed herein are also suitable deliverysystems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the likely mode of action of caPCNA-based peptideinhibitors and their roles in inhibiting cancer proliferation in thepresence of a DNA damaging agent.

FIG. 2 shows (A) cytotoxic effects of R9, caPeptide (126-133), andR9-caPep. U937 cells were treated with increasing amounts of eachpeptide and the level of apoptosis was evaluated by flow cytometry; and(B) Combined cytotoxicity of doxorubicin and R9-caPep. U937 cells weretreated with 25 μM R9-caPep and increasing concentrations ofdoxorubicin. The IC50 (thicker line) of doxorubicin decreases from 140nM to 110 nM when 25 μM of R9-caPep is added.

FIG. 3 shows nuclear localization of R9-caPep. U937 cells were treatedwith FITC-R9-caPep and collected on coated slides by cytospin.Fluorescent images for the DAPI stained nuclei (left) and FITC-R9-caPep(right) were overlaid (center) showing cellular uptake and nuclearlocalization of R9-caPep. Boxes indicate intact cells.

FIG. 4 shows structural interactions of FEN-1 with PCNA. Left: PCNAmonomers A and C with the molecular surface shown. aa330-380 of FEN-1monomers X and Z shown in wireframe representation. Right: Close-up ofthe region of strong hydrophobic interactions conserved in thePCNA/FEN-1 interaction. aa126 and aa133 form the edge of the cavitywhere FEN-1 interacts with PCNA.

FIG. 5 shows the interactions of aa126-133 peptide in the bindingpocket.

FIG. 6 shows orientation of amino acids 126-133 in the pocketsurrounding FEN-1. Portion of FEN-1 X and Z chains that fit into thecavity created by the molecular surface representation of PCNA. Positionof each amino acid from PCNA peptide 126-133 is indicated. Chart to theright lists the change in cytotoxicity when each amino acid issubstituted with an alanine.

FIG. 7 illustrates conserved residues of binding partners for PCNA.Left: Sequence conservation for various peptides/proteins and the fiveamino acid sequence which binds in the cavity partially created byaa126-133 of PCNA. Right: Overlay of the conserved sequences of the PCNAbinding partners shown in the cavity. The most conserved residues areindicated in both the table and the figure.

FIG. 8 illustrates a strategy behind the use of caPCNA peptides toinduce cell death in cancer cells. Mechanisms of caPCNA peptide ininhibiting PCNA action in cells without a critical DNA repair protein,BRCA1. caPCNA peptides are more effective in cells lacking BRCA1. WhenBRCA1 is present in tumors, they are often resistant to treatment by DNAdamaging agents like chemotherapy. Loss of BRCA1 often leads toaggressive tumors; however, the tumor cells are more sensitive to DNAdamaging agents since they lack this DNA repair protein.

FIG. 9 shows BRCA negative cells are more sensitive to the cytotoxicaction of caPCNA peptide.

FIG. 10 shows growth inhibition in HCC1937 cells lacking BRCA1 (A) andHCC1937 cells+BRCA1 (B) after 24-hr treatment with cisplatin orcisplatin plus R9-caPCNA peptide.

FIG. 11 shows R9-PCNA peptide lowers cisplatin dose needed for growthinhibition in HCC1937 cells lacking BRCA1 (A) and HCC1937 cells withBRCA1 (B) and is more effective in cells lacking BRCA1 (A). In HCC1937cells lacking BRCA1 (A), the IC₅₀ value for R9-PCNA peptide (60μM)+cisplatin is ˜20 μM and the IC₅₀ value for cisplatin alone is ˜155μM. In HCC 1937 cells with BRCA1 (B), the IC₅₀ value for R9-PCNA peptide(60 μM)+cisplatin is ˜100 μM and the IC₅₀ value for cisplatin aloneis >200 μM.

FIG. 12 shows that HCC1937 cells are more sensitive to cisplatin+caPCNApeptide combination treatment.

FIG. 13 shows summary of a 48-hr timepoint for EC50 values involvingcaPCNA peptides and cisplatin for HCC+ HCC− cells.

DETAILED DESCRIPTION

Methods and compositions disclosed herein relate to caPCNA-peptidevariants, peptidomimetics, functional analogs thereof that selectivelydisrupt vital cellular functions in cancer cells. There are at least twomodes of actions of these peptides. For example, caPCNA-derived peptidevariants either compete with caPCNA to bind to caPCNA-interactingproteins or alternatively bind to a site on caPCNA-interacting proteinthat disrupts the interaction.

Specific peptide variants derived from the caPCNA protein sequence havethe ability to block the binding of several cellular proteins thatparticipate in DNA replication, repair, cell cycle control, apoptosis,transcription, or chromosomal recombination in cancer cells. The bindingof caPCNA to these cellular proteins is disrupted when the peptide isallowed to compete with these proteins for their naturally occurringbinding site on PCNA. By disrupting the naturally occurring interactionbetween PCNA and the proteins that bind to or interact with PCNA, normalcellular functions that recruit PCNA are disrupted. This disruption ofvital cellular machinery renders the caPCNA-derived peptide variantscytotoxic by themselves or in combination with other molecules, such as,for example cancer chemotherapeutic drugs. These peptides, either aloneor in combination with other cancer therapy agents are useful cancerchemotherapeutics or augmentors of the pharmacodynamic effect ofspecific anti-cancer chemotherapeutics. The peptide inhibitors disclosedherein sensitize tumor cells towards chemotherapy agents that damage DNAand also render tumors that are generally resistant to cancer drugs moreresponsive.

Chemotherapeutic strategies are designed to exploit differences betweenmalignant versus nonmalignant cell biology with the overall goal beingselective inhibition of cancerous cells. Since, in many cases, cancercells rapidly proliferate in comparison to nonmalignant cells, they alsoexhibit an increase in (often erroneous) DNA repair processes. There arenumerous examples of drugs in the clinic that either induce DNA damageor inhibit DNA damage repair processes in malignant cells.

Chemotherapeutic agents such irradiation, doxorubicin, cisplatin, andthe like can be nonspecific, resulting in deleterious side effects inpatients. Without being bound by theory, it is believed herein thatcaPCNA peptides represent a novel strategy aimed at targeting caPCNA, acancer-associated isoform on which malignant cells rely for DNAreplication and repair processes.

The approximate IC₅₀s of the caPCNA peptide and its alanine-substitutedderivatives, P129A and Q131A, have been determined in HCC1937 andHCC1937+wild-type BRCA1 breast cancer cells. The ability of the caPCNApeptides to enhance the cytotoxic effects of DNA damaging agents isdescribed herein. A reduction of the IC₅₀ for cisplatin in the presenceof caPCNA peptides is discovered herein.

Lower doses of chemotherapeutic agents needed to kill cancer cells(reduce toxicity to patients without compromising efficacy) and increasesensitivity to chemotherapy in drug-resistant cells are useful intreating various cancer types.

Methods and compositions disclosed herein relate to caPCNA-peptidevariants, peptidomimetics, functional analogs thereof that selectivelydisrupt vital cellular functions in cancer cells. There are at least twomodes of actions of these peptides. For example, caPCNA-derived peptidevariants either compete with caPCNA to bind to caPCNA-interactingproteins or alternatively bind to a site on caPCNA-interacting proteinthat disrupts the interaction.

Without being bound by theory, it is believed herein that specificpeptide variants derived from the caPCNA protein sequence are able toblock the binding of one or more cellular proteins that participate inDNA replication, repair, cell cycle control, apoptosis, transcription,or chromosomal recombination in cancer cells. The binding of caPCNA tothese cellular proteins is disrupted when caPCNA derived peptides areallowed to compete with these proteins for their naturally occurringbinding site on PCNA. By disrupting the naturally occurring interactionbetween PCNA and the proteins that bind to or interact with PCNA, normalcellular functions that recruit PCNA are disrupted. This disruption ofvital cellular machinery renders the caPCNA-derived peptide variantscytotoxic by themselves or in combination with other molecules, such as,for example cancer chemotherapeutic drugs. These peptides, either aloneor in combination with other cancer therapy agents, are useful cancerchemotherapeutics or augmentors of the pharmacodynamic effect ofspecific anti-cancer chemotherapeutics. The peptide inhibitors disclosedherein sensitize tumor cells towards chemotherapy agents that damage DNAand also render tumors that are generally resistant to cancer drugs moreresponsive to treatments.

The term “sensitize” as used herein, for therapeutic purposes, generallyrefers to the ability of the peptides disclosed herein to lower theamount of a growth inhibitory agent or a cytotoxic agent (e.g.,doxorubicin, cisplatin, and the like) needed to kill 50% of a group ofcancer cells (e.g., a tumor) within a defined period of time (e.g., 24,48, 72 hours).

The terms “pre-cancerous” or “pre-malignant” generally refer to acondition which may or may not be recognizable as a morphological changein tissue architecture that is known to be, or thought to be, associatedwith the development of a cancer within that tissue (organ). Inaddition, the initial molecular changes in gene expression, (orexpression of specific isoforms of proteins), known to be associatedwith some percentage of cancer cells that are found in tumor tissues,may precede readily discernable morphological changes within the cellsof these tissues undergoing the cancer transformation process. Thus theinitial changes in the expression patterns of specific genes and/orproteins may be the first events associated with the moleculartransformation process leading to the development of a cancer; may onlybe recognizable at the molecular level, as they have not in themselvesinduced an alteration in the morphology of the cells, and/or tissue, tothe extent that it can be recognized by a trained individual(pathologist) at the light microscopic level.

The term “augment” or “augmenting” as used herein, for therapeuticpurposes, generally refers to an improvement in the pharmacodynamiceffect (referred to as the efficacy) of a therapeutic agent. Thus, theterm “augment” refers to the ability of the peptide to raise theefficacy of a therapeutic agent (e.g., doxorubicin, cisplatin, and thelike) leading to the killing of a greater number of cancer cells overthe same unit of time (e.g., 24, 48, or 72 hour period) when the peptideis administered prior to, along with, or after the therapeutic agent ascompared to the therapeutic agent alone.

Peptide variants derived from the protein Proliferating Cell NuclearAntigen (PCNA) are identified herein that have the ability to act, inconjunction with DNA damaging agents (e.g., doxorubicin), to enhance thetherapeutic effects of such agents to treat a variety of cancer cells.Without being bound by theory, it is believed herein that FIG. 1illustrates a mode of action of caPCNA-based peptide inhibitors andtheir roles in inhibiting cancer proliferation in the presence of a DNAdamaging agent. The peptides are derived from the amino acid sequencewithin PCNA, for example, encompassing amino acids 126-133 and includeone or amino acid mutations.

caPCNA-derived peptide variants and peptidomimetics represent novelanti-cancer therapeutic agents and also augment or sensitize tumor cellstowards existing cancer therapies.

Without being bound by theory, it is believed that the peptide sequencesdescribed herein target a region of the caPCNA protein that is likely tobe uniquely unfolded in cancer cells. Thus, the peptides disclosedherein are designed to selectively target tumor cells by virtue of theirability to compete with caPCNA for regulating the activity of specificproteins interacting with the amino acid sequences within PCNA that areinvolved in at least one of the following cellular processes: DNAreplication, repair, recombination, transcription, cell cycle checkpointcontrol, and apoptosis.

The peptides disclosed herein are synthesized using standard peptidesynthesis procedures and equipment or can be obtained commercially(e.g., United Biochemical Research Co., Seattle Wash.). A caPCNA-derivedpeptide that includes amino acids 126-133 of the human PCNA molecule(LGIPEQEY (SEQ ID NO: 1)) having at least one amino acid substitution,followed by a cell penetrating peptide (CPP) sequence, e.g., apolyarginine sequence to facilitate uptake of the peptide into cellsselectively inhibits cancer cells in vitro.

In certain embodiments involving in vitro methodologies, uptake ofcaPCNA peptide variants were initiated by incubation of this peptidewith the cancer cells in the presence of dimethyl sulfoxide (DMSO) ineither phosphate buffered saline (PBS) or culture media containing0.2-2% DMSO, without serum for about 4-24 hours. Uptake of thesepeptides is also efficiently mediated by encapsulation of the peptide ina liposome formulation and subsequent incubation with the cancer cellsat 37° C. for about 4-24 hours. These peptides also augment thecytotoxic effects of chemotherapeutic agents, such as doxorubicin,cisplatin, and the like.

The term “agent” as used herein includes nucleic acids, proteins,protein fragments, peptides, synthetic peptides, peptidomimetics,analogs thereof, small molecules, inhibitors, and any chemical, organicor bioorganic molecule capable of affecting protein-protein interactionor a cellular process.

The terms “caPCNA peptide variants” or “peptide variants” mean peptideswhose sequences were derived from caPCNA and include one or moremutations such as substitution mutations or deletion mutations or aminoacid analogs or a combination thereof. For example, LGIPEQEY (SEQ IDNO: 1) representing amino acids 126-133 of PCNA is caPCNA derivedsequence in which for example, amino acids G, P, Q, and penultimate Ecan be substituted with an amino acid such as alanine (A). Thus,LX₁IX₂EX₃X₄Y (SEQ ID NO: 35) is a peptide variant wherein X₁₋₄ can besubstituted either independently or collectively. In an embodiment, X₁is A, X₂ is P, X₃ is Q, and X₄ is E. In another embodiment, X₁ is G, X₂is A, X₃ is Q, and X₄ is E. In another embodiment, X₁ is G, X₂ is P, X₃is A, and X₄ is E. In another embodiment, X₁ is G, X₂ is P, X₃ is Q, andX₄ is A. In another embodiment, X₁ is G, X₂ is A, X₃ is A, and X₄ is A.The “caPCNA peptide variants” or “peptide variants” can range from about5-10, 5-50, 7-50, 8-20, 8-25, 8-30, 8-40, 8-50 amino acids in length.For example, the “caPCNA peptide variants” or “peptide variants” mayconsist essentially of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 contiguous amino acids fromcaPCNA wherein one or more amino acids are substituted. The “caPCNApeptide variants” or “peptide variants” may further include peptidetranslocation domains or sequences that enable the “caPCNA peptidevariants” or “peptide variants” to penetrate or translocate acrosscellular membranes. The “caPCNA peptide variants” or “peptide variants”are also be modified to affect their lipophilicity to enhance peptidedelivery into cancer cells. The peptides can be synthesized (“syntheticpeptides”) or can also be produced through recombinant techniques(“recombinant peptides”) or expressed in vivo using gene expressiontechniques. These peptides can also be engineered to increase their invivo stability (e.g., increase peptide stability by rendering themprotease resistant) without significantly affecting their efficacy ininhibiting caPCNA-protein interactions. Mutations including insertions,deletions, substitutions, amino acid modifications that substantially donot affect the inhibitory activity of the peptides disclosed herein arewithin the scope. Peptides that consist essentially of the 126-133sequence LGIPEQEY (SEQ ID NO: 1) having one or more mutations mayinclude other heterologous sequences that do not materially affect theinhibitory function of the peptide variants disclosed herein.

The peptides described herein show specificity for killing malignantcells compared to non-cancerous cells. For example, the peptidesdisclosed herein are substantially specific in which the peptidespreferentially kill malignant cancer cells more than 50%, preferablymore than 60% or 70% or 80% or 90% or 95% when compared to non-malignantcells.

A “peptide variant” or “peptide derivative” also refers to a moleculehaving an amino acid sequence of a region that is similar to a portionof PCNA or of a PCNA homolog, but additionally having at least onechemical modification of one or more of its amino acid side groups,α-carbon atoms, terminal amino group, or terminal carboxylic acid group.A chemical modification includes addition of chemical moieties, creationof new bonds, and/or removal of chemical moieties. Modifications atamino acid side groups include acylation of lysine, ε-amino groups,N-alkylation of arginine, histidine, or lysine, alkylation of glutamicor aspartic carboxylic acid groups, and deamidation of glutamine orasparagine. Modifications of the terminal amino include the des-amino,N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modificationsof the terminal carboxy group include the amide, lower alkyl amide,dialkyl amide, and lower alkyl ester modifications. A lower alkyl is aC1-C4 alkyl. Furthermore, one or more side groups, or terminal groups,may be protected by protective groups known to the ordinarily-skilledprotein chemist. The α-carbon of an amino acid may be mono- ordi-methylated.

Those of skill in the art recognize that peptides may be substantiallysimilar to the peptides described above in that an amino acid residuemay be substituted with another amino acid residue having a similar sidechain without substantially affecting the inhibitory functions of thepeptide variants disclosed herein. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidsubstitution groups include: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Thus, the peptide inhibitors disclosed herein mayhave one or more conservative amino acid substitutions withoutsubstantially affecting the inhibitory functions of the peptide.

Non-naturally occurring variants of the caPCNA-derived peptides canreadily be generated using recombinant techniques or chemical synthesis.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the disclosed peptides. Forexample, one class of substitutions is conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in the disclosed caPCNA-derived peptides by another amino acid oflike characteristics. Typically accepted as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu, and Ile; interchange of the hydroxyl residues Ser andThr; exchange of the acidic residues Asp and Glu; substitution betweenthe amide residues Asn and Gln; exchange of the basic residues Lys andArg; and replacements among the aromatic residues Phe and Tyr. A list ofpossible amino acid substitutions is provided in Table 5 herein.

The term “variant” refers to a peptide having an amino acid sequencethat differs to some extent from a native sequence peptide, that is anamino acid sequence that varies from the native sequence by conservativeamino acid substitutions, whereby one or more amino acids aresubstituted by another with same characteristics and conformationalroles. The amino acid sequence variants possess substitutions,deletions, and/or insertions at certain positions within the amino acidsequence of the native amino acid sequence.

The PCNA-derived peptide variants can also be fused or otherwise linkedto a ligand for a cell surface receptor that is present in cancer cells.The ligand is optionally cleavable such that the peptide variants(inhibitors) are targeted to tumor cells using a tumor-specific ligandbut are cleaved by a protease present in the tumor environment such thatthe peptide variants are free to enter the tumor cells. For example, thehuman transferrin receptor (hTfR), a marker for cellular proliferationis used as a target for therapeutics and is expressed at least 100-foldmore in oral, liver, pancreatic, prostate, and other cancers (Lee etal., (2001) “Receptor mediated uptake of peptides that bind the humantransferrin receptor” Eur. J. Biochem., 268: 2004-2012). Peptides,HAIYPRH (SEQ ID NO: 36) and THRPPMWSPVWP (SEQ ID NO: 37) bindspecifically hTfR and these peptides were able to target associatedmacromolecule to the hTfR (Lee, supra). These peptides bind sites thatdo not overlap with the native ligand, Tf, and are useful in vivo fortargeting macromolecules to the endocytic pathway in hTfR-positive cells(Lee, supra). Such peptides can also be used to target PCNA-derivedpeptides to enhance peptide delivery and also to further enhancespecific delivery.

The term “cell permeable factor” or “cell membrane carrier” or “cellpenetrating element” refers to any component including peptides thatenhance the ability of the peptide variants disclosed herein totranslocate the cell membrane as long as the factor does notsubstantially affect the ability of the peptide variants to inhibitcaPCNA interaction. Optionally, the cell-permeable factors operatethrough a non-endocytic and non-degradative pathway in mammalian cells.These factors may include cell penetrating peptides (CPP) or cellpermeable peptides. Examples of suitable cell-permeable peptides orpeptide domains to link or fuse caPCNA-derived peptides include, forexample, small polybasic peptides derived from the transduction domainsof certain proteins, such as polyarginine (R6-R21), the third-helix ofthe Antennapedia (Antp) homeodomain, an RYIRS (SEQ ID NO: 38) tagsequence, and those listed in Table 4 herein.

In an embodiment, the cell membrane permeable carrier is a peptide,preferably an arginine rich peptide. (see e.g., Futaki S. et al., (2001)“Arginine-rich peptides. An abundant source of membrane-permeablepeptides having potential as carriers for intracellular proteindelivery” J. Biol. Chem., 276, 5836). The number of arginine residues ina cell membrane permeable carrier peptide may contain more than 6arginines, preferably 7, 8, 9, 10, 11, 12, 13, 14, or 15 arginineresidues. The arginine residues in an arginine rich peptide need not becontiguous. One or more of the arginine residues may be a D-isomer ofarginine. Those of skill in the art know how to select an appropriatearginine rich peptide with a suitable number of arginine residues.

A peptide hairpin can also be used to make use of the increased numberof extracellular proteases surrounding tumor tissues (see US PatentApplication Publication 20070041904, incorporated herein by reference)to deliver the inhibitors (“cargo”) disclosed herein. The constructincludes a polyarginine peptide covalently attached to a polyanionicsegment, which would only be substantially internalized upon proteolyticcleavage of the anionic domain. Because the protease targeted is likelyoverexpressed on cancerous cells, internalization is more likely withthe tumor cells compared to a normal cell. Cellular association ofcell-penetrating peptides (CPPs) (e.g., polyarginine based) is blockedwhen they are fused to an inhibitory domain made up of negativelycharged residues. Such fusions termed as activatable CPPs (ACPPs)because cleavage of the linker between the polycationic and polyanionicdomains, usually by a protease, releases the CPP portion and itsattached peptide of interest (“cargo”) to bind to and enter cells suchas tumor cells.

Pretreatment of the cell with a polycation, cationic polymer and/orcationic peptide before transportation of the heterologous compound intothe cell is also useful (see e.g., U.S. Patent Application Publication20060083737, incorporated herein by reference).

Nuclear localization sequences (NLS) for example, VQRKRQKLMP (SEQ ID NO:39), SKKKKIKV (SEQ ID NO: 40), and GRKRKKRT (SEQ ID NO: 41) are alsouseful in transporting the peptide variants disclosed herein into tumorcells. Other NLS can be obtained for example at Nair et al., (2003),NLSdb: database of nuclear localization signals, Nucl. Acids Res.,31:397-399 (see also http://cubic.bioc.columbia.edu/db/NLSdb/).

In some embodiments, the peptide variants disclosed herein areconjugated to the cell membrane permeable carrier, optionally includinga spacer. For example, a polyarginine peptide having 5-9 arginineresidues may optionally include a non-arginine-based spacer peptide or aspacer having non-standard amino acids or amino acid analogs. Thespacers generally provide additional length to minimize for examplesteric hindrance to the function or transport of the peptide variantsdisclosed herein. Spacers may include about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15 or more amino acids. Suitable amino acids for use as spacersinclude for example, glycine.

The caPCNA peptide variants and the cell-permeable peptides are linkedby chemical coupling in any suitable manner known in the art as long asthe rendered conjugated proteins are biologically active. In an aspect,one way to increase coupling specificity is to directly chemicallycouple to a functional group found only once or a few times in one orboth of the polypeptides to be cross-linked. For example, in manyproteins, cysteine, which is the only protein amino acid containing athiol group, occurs only a few times. Also, for example, if apolypeptide contains no lysine residues, a cross-linking reagentspecific for primary amines will be selective for the amino terminus ofthat polypeptide. Successful utilization of this approach to increasecoupling specificity requires that the polypeptide have the suitablyrare and reactive residues in areas of the molecule that may be alteredwithout loss of the molecule's biological activity. Alternatively,synthetic peptides with a modified residue can be synthesized such thatspecificity of linking is enhanced.

Cysteine residues may be replaced when they occur in parts of apolypeptide sequence where their participation in a cross-linkingreaction would otherwise likely interfere with biological activity. Whena cysteine residue is replaced, it is typically desirable to minimizeresulting changes in polypeptide folding. Changes in polypeptide foldingare minimized when the replacement is chemically and sterically similarto cysteine. For these reasons, serine is preferred as a replacement forcysteine. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, whether thepolypeptide of interest is produced by chemical synthesis or expressionof recombinant DNA.

Coupling of the two constituents can be performed through a coupling orconjugating agent. Suitable intermolecular cross-linking reagentsinclude for example, J-succinimidyl 3-(2-pyridyldithio) propionate(SPDP) or N,N′-(1,3-phenylene)bismaleimide (both of which are highlyspecific for sulfhydryl groups and form irreversible linkages);N,N′-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11carbon methylene bridges (which relatively specific for sulfhydrylgroups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversiblelinkages with amino and tyrosine groups). Other cross-linking reagentsinclude: p,p′-difluoro-m,m′-dinitrodiphenylsulfon-e (which formsirreversible cross-linkages with amino and phenolic groups); dimethyladipimidate (which is specific for amino groups);phenol-1,4-disulfonylchloride (which reacts principally with aminogroups); hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate (which reacts principally with amino groups);glutaraldehyde (which reacts with several different side chains) anddisdiazobenzidine (which reacts primarily with tyrosine and histidine).

Cross-linking reagents may be homobifunctional, i.e., two functionalgroups that have the same reaction. A suitable homobifunctionalcross-linking reagent is bismaleimidohexane (“BMH”). Cross-linkingreagents may also be heterobifunctional. Heterobifunctionalcross-linking agents have two different functional groups, for examplean amine-reactive group and a thiol-reactive group, that will cross-linktwo proteins having free amines and thiols, respectively. Examples ofheterobifunctional cross-linking agents are succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide4-(p-maleimidophenyl)butyrate (“SMPB”), an extended chain analog of MBS.The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue. A hydrophilic moiety, such as a sulfonategroup, may be added to the cross-linking reagent to improve its watersolubility. Sulfo-MBS and sulfo-SMCC are examples of cross-linkingreagents modified for water solubility.

Many cross-linking reagents yield a conjugate that may be non-cleavableunder cellular conditions. However, some cross-linking reagents containa covalent bond, such as a disulfide, that is cleavable under cellularconditions. For example, Traut's reagent, dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl 3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavable cross-linkers. The use of acleavable cross-linking reagent permits the cargo moiety to separatefrom the transport polypeptide after delivery into the target cell.Direct disulfide linkage may also be useful.

Numerous cross-linking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincross-linking and conjugate preparation is: Wong, Chemistry of proteinconjugation and cross-linking, CRC Press (1991).

Chemical cross-linking may include the use of spacer arms. Spacer armsprovide intramolecular flexibility or adjust intramolecular distancesbetween conjugated moieties and thereby may help preserve biologicalactivity. A spacer arm may be in the form of a polypeptide moiety thatincludes spacer amino acids, e.g. proline. Alternatively, a spacer armmay be part of the cross-linking reagent, such as in “long-chain SPDP”(Pierce Chem. Co., Rockford, Ill., Cat. No. 21651 H).

Alternatively, the chimeric peptide can be produced as a fusion peptidethat includes the cell-permeable sequence and the caPCNA peptide variantsequence that can be expressed in known suitable host cells forlarge-scale production and purification. Fusion peptides, as describedherein, can be formed and used in ways analogous to or readily adaptablefrom standard recombinant DNA techniques, as described above.

Native peptides (in L-form) may be subject to degradation by naturalproteases, the peptides disclosed herein may be prepared to includeD-forms and/or “retro-inverso isomers” of the peptide. In this case,retro-inverso isomers of short fragments and variants of the peptide ofcaPCNA peptide variants disclosed herein are prepared. The caPCNApeptide variants can have one or more L-amino acids, D-amino acids, or acombination of both. For example, in various embodiments, the peptidesare D retro-inverso peptides. The term “retro-inverso isomer” refers toan isomer of a linear peptide in which the direction of the sequence isreversed and the chirality of each amino acid residue is inverted. See,e.g., Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature,368, 692-693 (1994). The overall result of combining D-enantiomers andreverse synthesis is that the positions of carbonyl and amino groups ineach amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. Unless specifically statedotherwise, it is presumed that any given L-amino acid sequence may bemade into a D retro-inverso peptide by synthesizing a reverse of thesequence for the corresponding native L-amino acid sequence.

Suitable chemotherapy agents include for example, cyclophosphamide(CYTOXAN™), capecitabine (XELODA™), chlorambucil (LEUKERAN™), melphalan(ALKERAN™), methotrexate (RHEUMATREX™), cytarabine (CYTOSAR-U™),fludarabine (FLUDARA™), 6-mercaptopurine (PURINETHOL™), 5-fluorouracil(ADRUCIL™), paclitaxel (TAXOL™), docetal, abraxane, doxorubicin(ADRIAMYCIN™), irinotecan (CAMPTOSAR™), cisplatin (PLATINOL™),carboplatin (PARAPLATIN™), oxaliplatin, tamoxifen (NOLVADEX™),bicalutamide (CASODEX™), anastrozole (ARIMIDEX™), examestane, letrozole,imatinib (GLEEVEC™), rituximab (RITUXAN™), trastuzumab (HERCEPTIN™),gemtuzumab, ozogamicin, interferon-alpha, tretinoin (RETIN-A™, AVITA™,RENOVA™), arsenic trioxide, bevicizumab (AVASTIN™), bortezombi(VELCADE™), cetuximab (ERBITUX™), erlotinib (TARCEVA™), gefitinib(IRESSA™), gemcitabine (GEMZAR™), lenalidomide (REVLIMID™), Serafinib,Sunitinib (SUTENT™), panitumumab (VECTIBIX™), pegaspargase (ONCASPAR™),and Tositumomab (BEXXAR™) and prodrugs or precursors or combinationsthereof.

Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxanes, and topoisomerase IIinhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, andbleomycin. Those agents that arrest G1 also spill over into S-phasearrest, for example, DNA alkylating agents such as tamoxifen,prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate,5-fluorouracil, and ara-C.

In an embodiment, the dosage of chemotherapy agents, when used inconjunction with the peptides of the disclosure, may be lower than thedosage used for a monotherapy. For example, doxorubicin, cisplatin, andthe like when coadministered (either before or during or after) withcaPCNA peptides described herein, the dosage may be lowered by 25% or35% or 50% or 60% or 75% or 80% of the standard dose. Depending on otherfactors, dosage may range from 300 mg/m² to 500 mg/m². Other suitabledoses may be lower e.g., 20 mg/m², 50 mg/m², 100 mg/m², 150 mg/m², or200 mg/m² or higher 400 mg/m², 450 mg/m², 500 mg/m², 550 mg/m², or 600mg/m².

The peptides disclosed herein are also suitable for cancer patientsundergoing radiotherapy (including all forms of ionizing radiations thatare DNA damaging) and any other forms of cancer therapy. The amount ofradiation used in radiation therapy is measured in gray (Gy), and mayvary depending on the type and stage of cancer being treated. Forcurative cases, a typical dose for a solid epithelial tumor ranges fromabout 60 to 80 Gy, while lymphoma tumors are treated with about 20 to 40Gy. Preventative (adjuvant) doses of radiation are typically around45-60 Gy in 1.8-2 Gy fractions (e.g., for breast, head and neck cancersrespectively.) Many other factors are considered by radiationoncologists when selecting a dose, including whether the patient isreceiving chemotherapy or any other therapy, whether radiation therapyis being administered before or after surgery, and the degree of successof surgery. A typical fractionation schedule for adults is about 1.8 to2 or to about 3 Gy per day. If the peptides of the present invention areused in combination with radiotherapy, then the radiation doses mayreduced by, for example, 10%, 20%, 30%, 40%, 50%, 60%, and 75%. Modes ofdelivering radiotherapy include for example, conventional external beamradiotherapy (2DXRT), 3-dimensional conformal radiotherapy (3DCRT),stereotactic radiotherapy, image-guided radiation therapy (IGRT), andintensity-modulated radiation therapy (IMRT).

Particle therapy (Proton therapy) that uses energetic ionizing particles(protons or carbon ions) is also suitable. Radioisotope therapy (RIT)includes the use of radioisotopes to target tumor tissues. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu.

Radioimmunotherapy includes the use of biologicals such as antibodiesand a radioisotope. Ibritumomab tiuxetan (Zevalin™) is a monoclonalantibody anti-CD20 conjugated to a molecule of Yttrium-90. TositumomabIodine-131 (Bexxar™) is a molecule of Iodine-131 linked to themonoclonal antibody anti-CD20.

The peptide inhibitors disclosed herein are suitable augmenting agentsthat can be administered either prior to, during, and afteradministering a particular cancer therapy, e.g., chemotherapy orradiotherapy.

A “small molecule” refers herein to have a molecular weight below about500 Daltons.

It is to be understood that cancers suitable for treatment using thepeptides disclosed herein include, but are not limited to, malignanciessuch as various forms of glioblastoma, glioma, astrocytoma, meningioma,neuroblastoma, retinoblastoma, melanoma, colon carcinoma, lungcarcinoma, adenocarcinoma, cervical carcinoma, ovarian carcinoma,bladder carcinoma, lymphoblastoma, leukemia and other blood cancers,osteosarcoma, breast carcinoma, hepatoma, nephroma, adrenal carcinoma,or prostate carcinoma, esophageal carcinoma. If a malignant cellexpresses caPCNA isoform, the compositions disclosed herein are capableof disrupting the interaction of caPCNA isoform with one or moreproteins. Metastases of cancers are also treated by the peptideinhibitors disclosed herein. Any cell, whether cancerous or premalignantor precancerous, if it expresses cancer specific PCNA isoform, issuitable for reducing cellular proliferation or chemoprevention.

Non-peptidic compounds that mimic peptide sequences are known in the art(Meli et al. J. Med. Chem., 49:7721-7730 (2006), that describes methodsof identifying nonpeptide small molecule mimics). Synthesis ofnon-peptide compounds that mimic peptide sequences is also known in theart (see, e.g., Eldred et al. J. Med. Chem., 37:3882, (1994); Ku et al.J. Med. Chem., 38:9, (1995); Meli et al. J. Med. Chem., 49:7721-7730(2006)). Such nonpeptide compounds that mimic caPCNA-derived peptides orvariants thereof disclosed herein that bind caPCNA are contemplated bythe present invention.

The term “peptidomimetic” or “peptide mimetic” refers to a chemicalcompound having small protein-like chain (peptide) that includesnon-peptidic elements such as non-natural amino acids. Peptidomimeticsare designed and synthesized with the purpose of binding to targetproteins in order to induce or effect a particular change. Generally, apeptidomimetic functions by mimicking or antagonizing key interactionsof the parent peptide structure that it was designed to mimic orantagonize. A peptidomimetic normally does not have classical peptidecharacteristics such as enzymatically cleavable peptidic bonds. For ageneral review of the various techniques available for design andsynthesis peptide mimetics, see al-Obeidi et al., (1998), “Peptide andpeptidomimetic libraries. Molecular diversity and drug design” MolBiotechnol.; 9(3):205-23; and Houben-Weyl: Synthesis of Peptides andPeptidomemetics, Thieme Medical Publishers, 4^(th) edition (2003).

As used herein, the terms “peptide mimetic,” “peptidomimetic,” and“peptide analog” are used interchangeably and refer to a syntheticchemical compound that has substantially the same structural and/orfunctional characteristics of caPCNA peptides or variants thereofdisclosed herein. The mimetic can be either entirely composed ofsynthetic, non-natural analogues of amino acids, or, is a chimericmolecule of partly natural peptide amino acids and partly non-naturalanalogs of amino acids. The mimetic can also incorporate any amount ofnatural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or inhibitory or binding activity. Routine experimentation willdetermine whether a mimetic is within the scope of the disclosure, i.e.,that its structure and/or function is not substantially altered. Thus, amimetic composition is within the scope if it is capable of specificallyinhibiting caPCNA-mediated cellular proliferation or cell death.

Polypeptide mimetic compositions can contain any combination ofnonnatural structural components, which are typically from threestructural groups: a) residue linkage groups other than the naturalamide bond (“peptide bond”) linkages; b) non-natural residues in placeof naturally occurring amino acid residues; or c) residues which inducesecondary structural mimicry, i.e., to induce or stabilize a secondarystructure, e.g., a beta turn, gamma turn, beta sheet, alpha helixconformation, and the like.

A polypeptide can be characterized as a mimetic when all or some of itsresidues are joined by chemical means other than natural peptide bonds.Individual peptidomimetic residues can be joined by peptide bonds, otherchemical bonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N=dicyclohexylcarbodiimide (DCC) or N,N=diisopropylcarbodiimide (DIC).Linking groups that can be an alternative to the traditional amide bond(“peptide bond”) linkages include, e.g., ketomethylene (e.g., —C═O—CH₂for —C═O—NH—), aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether(—CH₂—O), thioether (CH₂—S), tetrazole (CN₄), thiazole, retroamide,thioamide, or ester (see, e.g., Spatola (1983) in Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357,A Peptide Backbone Modifications, Marcell Dekker, NY).

A polypeptide can also be characterized as a mimetic by containing allor some non-natural residues in place of naturally occurring amino acidresidues. Nonnatural residues are well described in the scientific andpatent literature; a few exemplary nonnatural compositions useful asmimetics of natural amino acid residues.

In another embodiment, peptides capable of disrupting caPCNA interactioninclude peptides of amino acid sequences with one or more amino acidsubstitutions that include about +3 contiguous or non contiguousadditional amino acids on the NH₂ terminus of LGIPEQEY (SEQ ID NO: 1)and about +9 contiguous or non contiguous amino acids on the COOHterminus of LGIPEQEY (SEQ ID NO: 1). For example, some of these peptidesinclude amino acid sequences of QLGIPEQEYSC (SEQ ID NO: 21) (+1-NH2terminus, +2-COOH terminus), VEQLGIPEQEY (SEQ ID NO: 22) (+3-NH2terminus), LGIPEQEYSCVVK (SEQ ID NO: 23) (+5-COOH terminus),LGIPEQEYSCVVKMPSG (SEQ ID NO: 24) (+9-COOH terminus), EQLGIPEQEY (SEQ IDNO: 25) (+2-NH2 terminus), QLGIPEQEY (SEQ ID NO: 26) (+1-NH2 terminus),LGIPEQEYSCVVKMPS (SEQ ID NO: 27) (+8-COOH terminus), LGIPEQEYSCVVKMP(SEQ ID NO: 28) (+7-COOH terminus), LGIPEQEYSCVVKM (SEQ ID NO: 29)(+6-COOH terminus), LGIPEQEYSCVV (SEQ ID NO: 30) (+4-COOH terminus),LGIPEQEYSCV (SEQ ID NO: 31) (+3-COOH terminus), LGIPEQEYSC (SEQ ID NO:32) (+2-COOH terminus), QLGIPEQEYSC (SEQ ID NO: 33) (+1-NH2 terminus,+2-COOH terminus), LGIPEQEYS (SEQ ID NO: 34) (+1-COOH terminus) andcombinations of the additional NH₂ and COOH termini amino acids thatflank LGIPEQEY (SEQ ID NO: 1). Amino acid mutations includingsubstitutions that do not affect the specificity of the peptides togenerate csPCNA specific antibodies are within the scope of thisdisclosure. One or more of the amino acid residues in the peptides maybe replaced with an amino acid analog or an unnatural or non-standardamino acid.

Dosage of the caPCNA-derived peptide variants depends on the efficacy ofthe peptides, stability of the peptides in vivo, mode of administration,the nature of cancer being treated, body weight, age of the patient andother factors that are commonly considered by a skilled artisan. Forexample, dosage of caPCNA-derived peptide variants drug can range fromabout 0.1-10.0 microgram (mcg)/kg body weight or from about 0.2-1.0mcg/kg body weight or from about 0.5-5.0 mcg/kg body weight or fromabout 10.0-50.0 mcg/kg body weight. Depending on the toxicity effectsand tumor killing capability, the dosage can also range from about1.0-10.0 mg/kg body weight and from about 0.1-1.0 mg/kg body weight. Theamount of the inhibitor that is administered to the subject can and willvary depending upon the type of inhibitor, the subject, and theparticular mode of administration. Those skilled in the art willappreciate that dosages may also be determined with guidance fromGoodman & Goldman's The Pharmacological Basis of Therapeutics, NinthEdition (1996), Appendix II, pp. 1707-1711 and from Goodman & Goldman'sThe Pharmacological Basis of Therapeutics, Tenth Edition (2001),Appendix II, pp. 475-493.

Administration of the compositions disclosed herein may be via any routeknown to be effective by the physician of ordinary skill Parenteralroutes include intravenous, intramuscular, subcutaneous, andintraperitoneal routes of administration. Intravenous, intramuscular,and subcutaneous routes of administration of the compositions disclosedherein are suitable. For parenteral administration, the peptidesdisclosed herein can be combined with phosphate buffered saline (PBS) orany suitable pyrogen-free pharmaceutical grade buffer that meets FDAstandard for human subject administration. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,diluents, or other liquid vehicle, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, 20^(th) Edition, A.R. Gennaro (Williams and Wilkins, Baltimore, Md., 2000) disclosesvarious carriers used in formulating pharmaceutical compositions andknown techniques for the preparation thereof. Solutions or suspensionsof the compositions described herein can also include a sterile diluent,such as water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propyleneglycol or other synthetic solvents;chelating agents, such as EDTA; buffers, such as acetates, citrates orphosphates; and agents for the adjustment of tonicity, such as sodiumchloride or dextrose. A parenteral preparation of the compositions canbe enclosed in ampoules, disposable syringes or multiple dose vials madeof glass or plastic, in accordance with standard practice in the field.The compositions disclosed herein can be stored as a lyophilized sterilepowder in vials containing for reconstitution and the unreconstitutedproduct may be stored at −20° C.

Agents administered parenterally, i.e., intravenously, intramuscularly,etc., may include a sterile diluent such as water, saline solution, apharmaceutically acceptable polyol such as glycerol, propylene glycol,polyethylene glycols, or other synthetic solvents; an antibacterialand/or antifungal agent such as benzyl alcohol, methyl paraben,chlorobutanol, phenol, thimerosal, and the like; an antioxidant such asascorbic acid or sodium bisulfite; a chelating agent such asetheylenediaminetetraacetic acid; a buffer such as acetate, citrate, orphosphate; and/or an agent for the adjustment of tonicity such as sodiumchloride, dextrose, or a polyalcohol such as mannitol or sorbitol. ThepH of the solution may be adjusted with acids or bases such ashydrochloric acid or sodium hydroxide. Preparations for oraladministration generally include an inert diluent or an edible carrier.They may be include a pharmaceutically compatible binding agent such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; and/or a flavoring agent such aspeppermint, methyl salicylate, or citrus flavoring. Oral preparationsmay be enclosed in gelatin capsules, compressed into tablets, orprepared as a fluid carrier. For administration by inhalation, the agentis generally delivered in the form of an aerosol spray from apressurized container or dispenser that contains a suitable propellant,e.g., a gas such as carbon dioxide, or a nebulizer. For topical (e.g.,transdermal or transmucosal) administration, penetrants appropriate tothe barrier to be permeated are generally included in the preparation.Transmucosal administration may be accomplished through the use of nasalsprays or suppositories, and transdermal administration may be viaointments, salves, gels, patches, or creams as generally known in theart.

Peptides and other compositions disclosed herein can be administered viaany suitable means. For example, the peptide compositions may be dilutedin saline or any suitable buffer and administered directlyintravenously. For example, the peptide compositions can be encapsulatedin liposomes and administered intravenously of by any suitable method.For example, the peptide compositions can be delivered by an extendedrelease drug delivery system known to one of ordinary skill in the art.Other modes of targeting tumors are also suitable. For example, U.S.Patent Application Publication US20050008572 (Prokop et al.,) disclosesmethods and compositions relating to nanoparticular tumor targeting andtherapy, the disclosure of which is hereby incorporated by reference.U.S. Patent Application Publication US20030212031 (Huang et al.,)discloses stable lipid-comprising drug delivery complexes and methodsfor their production, the disclosure of which is hereby incorporated byreference.

Replication defective viral expression vectors (e.g., lentivirus;adenovirus, adeno-associated virus, herpes virus, and others) capable ofexpressing the peptides disclosed herein are also suitable deliverysystems. Other nucleic acid delivery systems such as retroviral vectors,adenovirus vectors, adeno-associated virus vectors, alphavirus vectors,Semliki Forest virus-based vectors, Sindbis virus-based vectors are alsouseful, See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol.5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.

caPCNA-derived peptides disclosed herein are expressed from a suitableviral vector. cDNA sequence of PCNA gene is found for example, inTravali et al., (1989), J. Biol. Chem. 264 (13), 7466-7472, incorporatedby reference and also in several GenBank entries such as for example,NM_(—)002592 and NM_(—)182649. Standard cloning methods based on PCR andsite-directed mutagenesis techniques are used to engineer one or moreAla substations or other conserved mutations in the coding region ofcaPCNA regions for expressing the variant peptides in a cancer cell.

In addition, the term “pharmaceutically effective amount” or“therapeutically effective amount” refers to an amount (dose) effectivein treating a patient, having, for example, breast cancer. For example,in vitro test conditions, a suitable dose would kill at least 50% ofcancer cells. It is also to be understood herein that a“pharmaceutically effective amount” may be interpreted as an amountgiving a desired therapeutic effect, either taken in one dose or in anydosage or route, taken alone or in combination with other therapeuticagents. In the case of the present disclosure, a “pharmaceuticallyeffective amount” may be understood as an amount of caPCNA-derivedpeptide variants which may for example, suppress (e.g., totally orpartially) the interaction of caPCNA and one or more of its interactingpartners or reduce tumor growth or reduce cancer cell proliferation.

The following examples are exemplary and are not intended to limit thescope of the disclosure.

Example 1 Cytotoxic Effects of Cell-Permeable caPCNA-Derived PeptideVariants on Breast Cancer Cells

caPCNA derived peptides (Table 1 and Table 2) introduced into breastcancer cells compete with PCNA for binding of its partner protein and,thereby, prohibit full-length PCNA from interacting with them. Thispeptide represents part of the inter-domain connecting loop (IDCL) ofPCNA and competes with PCNA for binding to proteins such as polymeraseδ, xeroderma pigmentosum group G (XPG), or FEN-1 which are necessary tocarry out proper DNA replication and repair. A decrease in a cell'sability to properly replicate and repair its DNA would eventually leadto apoptotic cell death. This is likely to lead to dysfunctional DNAreplication and repair, resulting in the death of cancer cells.

A nonaarginine-linked caPCNA derived peptide (R9-caPep) was used to testthe effect of disrupting caPCNA interaction. One group ofcell-penetrating peptides relies on positively charged amino acids forcellular uptake. Polyarginines have been shown to be more efficient thanpolylysines and polyhistidines at promoting cellular uptake.Specifically, D-arginine sequences are more efficient than L-argininesequences. Thus, a D-nonaarginine linked caPeptide was created with thesequence RRRRRRRRRCCLGIPEQEY (R9-caPep).

U937 monocytic lymphoma cells grow in suspension and have been shown tobe a model cell line to use in therapeutic peptide evaluation since thecell line grows very quickly and provides highly reproducible results.Western blot analysis indicated that this cell line contains both theacidic (caPCNA) and basic (nmPCNA) isoforms of PCNA. First, it wastested whether or not the R9 sequence alone had any cytotoxic effect inU937 cells. The U937 cells were treated with up to 100 μM of the R9sequence for 24 hours. The R9 sequence did not promote apoptosis inthese cells (FIG. 2A). Cells were then treated with increasingconcentrations of unlinked caPeptide to ensure that it did not possesscytotoxic activity without the N-terminal arginine sequence. Up to 200μM of caPeptide was added to the U937 cells for 24 hours. caPeptidealone did not have any cytotoxic activity (FIG. 2A).

To test cellular uptake and nuclear localization of R9-caPep, anN-terminal fluorescein isothiocyanate (FITC) residue was attached toallow microscopic visualization of the peptide's cellular location. TheU937 cells were treated with 10 μM of FITC-R9-caPep for 24 hours. Thecells were then collected onto a poly-L-lysine coated slide by cytospinand mounted with a media containing the DNA staining dye4′-6-Diamidino-2-phenylindole (DAPI), which complexes with doublestranded DNA and indicates the location of the nucleus. A Leica DM500fluorescent microscope was used to visualize the staining. FIG. 2B 4shows the DAPI stained nuclei (blue) and the FITC labeled R9-caPep(green) do overlap indicating that the R9-caPep was internalized by thecells and was accessible to the nucleus.

Once it was established that the R9 sequence promoted cellular uptake ofthe peptide, increasing amounts of R9-caPep were added to U937 cells todetermine if the peptide's uptake would have any cytotoxic effect.R9-caPep concentrations above 20 μM were observed to induce a markedincrease in apoptotic cells with almost 93% of cells being apoptotic inthe presence of 50 uM of R9-caPep (FIG. 2A).

See FIG. 3 that shows nuclear localization of R9-caPep. U937 cells weretreated with FITC-R9-caPep and collected on coated slides by cytospin.Fluorescent images for the DAPI stained nuclei (left) and FITC-R9-caPep(right) were overlaid (center) showing cellular uptake and nuclearlocalization of R9-caPep. Boxes indicate intact cells.

To verify that the cytotoxicity of the R9-caPep was due to the PCNApeptide sequence itself, a scrambled peptide using the same eight aminoacids, R9-Scrambled, was prepared (Table 3). Treatment of U937 cellswith R9-Scrambled resulted in little cytotoxicity (Table 3). These dataindicated that the cytotoxicity of the R9-caPep was due to the caPCNApeptide sequence and was not due to any potential cellular damage fromuptake of peptide.

caPeptide was incubated with U937 cells for 24 hours but no increase inapoptosis was detected. Since most peptides are not passivelytransferred into cells, a nonaarginine linker was attached to theN-terminal portion of caPeptide to create R9-caPep. Fluorescentvisualization of the R9-caPep revealed that cellular uptake occurs andthat the peptide can locate to the nucleus. The R9 sequence alone wastested for cell toxicity and it did not cause an increase apoptosis.However, R9-caPep caused a marked increase in apoptosis, especially atconcentrations above 20 μM with almost 100% cell death at 50 μM. Whenthe amino acid sequence of caPeptide was scrambled and attached to theR9-linker to create R9-Scrambled, no cytotoxicity was detected.

Further as shown in Table 6, the caPCNA inhibiting peptide wassubstantially specific in killing cancer cells as compared tonon-malignant cells. In order to demonstrate that the work with U937cells was directly relatable to our breast cancer studies, the relativesensitivity of a breast cancer cell line (MCF7) and a non-malignantbreast cell line (MCF-10A) to the R9-caPeptide was determined. TheR9-caPeptide was evaluated for cytotoxic effects in non-malignant (MCF10A) and malignant (MCF 7) breast cells (Table 6). The cells weretreated with 50 μM R9-caPeptide for 48 hours and the extent of celldeath was evaluated by flow cytometry. R9-caPeptide was observed to beapproximately five times more cytotoxic for malignant breast cells(MCF7) than non-malignant breast cells (MCF10A). The significant levelof cytotoxicity in breast cancer cells is likely due to enhanced bindinginteraction of caPeptide with specific DNA repair proteins in thesecells, as indicated for example by the observed preferential binding ofcaPCNA for XPG protein, leading to an inhibition of DNA repair in thesecells, and ultimately cell death.

Therefore, this example demonstrated that R9-caPep selectively disruptedthe caPCNA interaction with one or more of its binding partners andresulted in cell death.

Example 2 R9-caPep Augments the Action of Doxorubicin and SensitizesTumor Cells for Chemotherapy

Since R9-caPep was effective at killing cancer cells, it was testedwhether combining the peptide with a DNA damaging chemotherapeutic agentwould allow for a lower concentration of the drug to be used.Doxorubicin is a DNA damaging agent used to treat many types of solidtumors and acute leukemias. It is a very effective drug but one of itsmain side effects is cardiomyopathy. Due to the damage that doxorubicincauses to the heart, patients are only allowed to receive a totallifetime dose of less than 500 mg/sq m. If lower doses of doxorubicin,combined with a cytotoxic dose of R9-caPep, can cause the same amount ofcell death as high concentrations of doxorubicin alone, it would bepossible to use less doxorubicin which would likely reduce harmful sideeffects. U937 cells were treated with 25 μM of R9-caPep together withincreasing concentrations of doxorubicin for 24 hours. The combinationof R9-caPep and doxorubicin reduces the median inhibitory concentration50 (IC₅₀) of doxorubicin from approximately 140 nM to 110 nM (FIG. 2B).The effect that R9-caPep has in combination with doxorubicin seems to bemore pronounced at lower concentrations. When the U937 cells are treatedwith 100 nM of doxorubicin 30% cell death is achieved; however, only 25nM of doxorubicin is needed to achieve the same level of cytotoxicitywhen combined with 25 μM of R9-caPep. Although there is not a largereduction in the IC₅₀ when 25 μM of R9-caPep is administered incombination with doxorubicin, any reduction in the amount of doxorubicinneeded could potentially reduce the harmful side effects the drug wouldhave on a patient.

Cell types (e.g., breast, colon, lung, leukemias, and the like)expressing the caPCNA isoform are sensitive to the caPCNA-derivedpeptides, as these peptides' core structure resemble the a uniquestructure within the caPCNA isoform (and absent from the non-malignant(nm) PCNA isoform due to the lack of post-translational modification ofone or more of the acidic amino acids in a stretch of 8 amino acids(126-133) in the caPCNA isoform. The sensitivity of leukemia cells andneuroblastoma cells to these peptides were tested. Colon cancer cells,cervical cancer cells, ovarian cancer cells, prostate cancer cells, lungcancer cells, esophageal cancer cells, liver cancer cells, pancreaticcancer cells, breast cancer cells, melanoma cancer cells, (and otherforms of cancer) all express the caPCNA isoform. Thus, all of thesecancer types and any other cancer type that expresses caPCNA aresensitive to the cytotoxic effects of the caPCNA-derived peptides orvariants thereof on these cancer cells.

Thus, R9-caPep (or any other caPCNA-based peptides or variants thereofdisclosed herein) is useful as a therapeutic agent either alone or incombination with other DNA damaging agents. The harmful side effectsthat result from treatment with chemotherapeutic agents like doxorubicinis substantially reduced if the drug is used in combination withR9-caPep. This example demonstrates that R9-caPep is capable ofaugmenting the cytotoxic effects of a DNA damaging agent and also likelyrenders resistant tumors sensitive to cancer therapy agents andradiation therapy.

Example 3 Identification of Interacting Amino Acids for the CytotoxicAction of R9-caPep

The cancer associated epitope of PCNA is also part of the flexible IDCLregion of PCNA. This region of PCNA is considered to have a disorderedstructure. Disordered regions in proteins are usually associated withprotein-to-protein interaction sites. The cytotoxic effect of R9-caPepindicates that it interacts with full length PCNA's binding partners andinhibiting the normal processes that occur via PCNA binding. Todetermine which amino acids in the R9-caPep sequence are necessary forbinding of the peptide to these proteins, peptide constructs weregenerated that mutated each amino acid individually to an alanine (Table3). Each alanine mutant peptide of caPCNA was used to treat U937 cellsfor 24 hours. An amino acid that is important to protein binding wouldpresumably show decreased cell cytotoxicity when it is substituted withan alanine. Those amino acids which are not critical for binding couldbe anticipated to show a cytotoxic level similar to that of theunsubstituted peptide. For a comparison of the potential cytotoxiceffect of each of the alanine substituted R9-linked caPeptides, U937cells were treated with 30 μM of peptide. This was the concentration atwhich R9-caPep resulted in almost 50% cell death (FIG. 2A). The resultsof this study are shown in Table 3. It was observed that peptidescontaining an alanine substituted at position aa126 or aa133 show littlecytotoxic activity and therefore were likely important for the peptide'sactivity. The peptide with an alanine substituted at amino acid position128 exhibited a decrease in cytotoxicity though not to the extentobserved with substitutions at positions 126 or 133. Surprisingly,alanine substitutions at amino acid positions 129, 131, or 132 wereobserved to significantly increase the cytotoxic activity of thepeptide.

Thus, this example demonstrates that amino acid substitutions to caPCNAderived peptide provide various peptide variants of therapeutic use.

Example 4 Molecular Modeling of the Observed caPeptide AlanineSubstituted Effects on Cytotoxicity

To gain insight into the potential relationships between the observedcytotoxic effects of the alanine substituted caPeptides and theirability to affect the recognition of the alternated peptide by PCNApartners, in silico molecular modeling studies were performed. Toperform these studies, the interaction of PCNA and its known bindingpartner FEN-1 were modeled. FEN-1 is one of the few PCNA bindingpartners for which the x-ray crystal structure is actually known and,therefore, would facilitate the modeling study. The crystal structure ofthe PCNA homotrimer bound to three FEN-1 molecules was developed.

Each FEN-1 molecule binds to its corresponding PCNA monomer in adifferent orientation with PCNA molecules A, B, and C binding to FEN-1molecules X, Y, and Z respectively. The difference in the orientation ofeach FEN-1 monomer is clearly shown when corresponding PCNA/FEN-1monomer pairs A-X and C-Z are overlaid. However, the C-terminal tailregions of both the X and Z chains of FEN-1 (aa330-380) interact withIDCL portion of PCNA. (see FIG. 5). When this complex was rotated 90°and the PCNA molecule was depicted in a space-filling model, it becameapparent that the aa126-133 peptide formed a cavity that fits aroundFEN-1 amino acid residues that interacted in the area of the cavity(FIG. 6). This cavity was flanked by aa126 and aa133 of PCNA, the aminoacids which were shown to be important for the cytotoxic activity ofR9-caPep by the alanine mutation analysis (Table 3). A closer look atthe amino acid R groups of aa126-133, in relation to the cavity, revealsthat aa126, aa128, and aa133 face the cavity and provide contact pointsfor the PCNA/FEN-1 interaction (FIG. 7). However, the R groups of aa127and aa130 point away from the pocket and do not appear to be involved inPCNA/FEN-1 binding (FIG. 7). Of the amino acids that were shown toenhance cytotoxic activity by the alanine mutation analysis, aa129 andaa131 face the cavity and aa132 points away from the cavity. When theseamino acids are mutated to an alanine, a conformational change occurswhich may cause the cavity to close more tightly around the bindingpartner thereby creating a stronger interaction.

Although FEN-1 had been used as the model for this study, the cavityformed by aa126-133 is also important for binding of PCNA to many of itspartners. FIG. 7 lists several PCNA binding partners and the conservedsequences used to interact with PCNA. When the five amino acid consensusbinding sequence of each peptide or protein was fit into the PCNAspace-filling model where it is reported to interact, they all overlayin the pocket region formed by caPeptide. Although there are many otheramino acid interacting points between PCNA and its binding partners, thecavity formed by the caPeptide region may be necessary for properbinding and the initiation of the cellular processes produced by theinteraction.

In silico molecular modeling was performed to further investigate thecontribution of the PCNA amino acid sequence to R9-caPep cytotoxicity.The positions of aa126-133 were determined from the crystal structure ofPCNA bound to FEN-1. The analysis revealed that aa126-133 of PCNA form acavity in which a conserved five amino acid sequence of several ofPCNA's binding partners fit. The amino acids determined to be importantfor the activity of R9-caPep from the alanine mutation analysis were thesame amino acids which face the cavity and possibly provide contactpoints between PCNA and the binding partner (aa126, aa128, and aa133).Those amino acids that are not important for the cytotoxic activity ofR9-caPep face away from the pocket and do no appear to participate inthe binding of PCNA to its partner (aa127 and aa130). Of the amino acidsthat cause an increase in cytotoxicity, aa129 and aa131 face the pocketand aa132 is turned away from the pocket.

The cellular cytotoxicity shown by R9-caPep is thought to be caused bythe peptide acting as decoy for PCNA's binding partners. Since thepeptide mimics the IDCL of full length PCNA, it could bind to proteinssuch as XPG, polymerase δ, and FEN-1 thus preventing them from bindingto intact PCNA. Sequestration of these proteins by the peptide wouldprevent proper DNA replication and repair and lead to cell death.Mutation of certain amino acids (aa126, aa128, and aa133) of R9-caPepdecreases its ability to be recognized by PCNA's binding partners andreduces the peptides ability to disrupt PCNA-partner interactions thusleading to decreased cell death. The mutation of amino acids 129, 131,and 132 to an alanine may cause a conformational change which allows thepeptide to bind more tightly to potential partners thus causingincreased cytotoxicity.

Example 5 Chemoprevention Using caPCNA Peptides and Variants Thereof

caPCNA peptides and variants thereof are used as chemo-preventativeagents for chemoprevention, in which the peptides are effective inlimiting the transformation of, for example, pre-cancerous cells intocancerous cells or detectable tumors. Due to the ability of the caPCNAderived peptides or the variants thereof to selectively kill cancercells while having a minimal effect on the viability of non-cancercells, these peptides or the variants are useful in preventingcarcinogenesis. This approach is based on the observation that becausethe expression of the cancer specific isoform of PCNA (caPCNA) is arelatively early event in the transformation process of cells leading toa malignant phenotype, blocking the interaction of caPCNA with one ormore of its binding partners at an early stage, results in an effectivechemoprevention.

Any tissue that is capable of expressing caPCNA isoform at an earlystage prior to the development of a detectable tumor mass or a malignantphenotype, is suitable for chemopreventive therapy using the caPCNApeptides and the variants thereof disclosed herein. Depending on thetoxicity of the caPCNA derived peptides and the variants disclosedherein, a skilled artisan would readily recognize and optimize thedosage ranges to implement an effective chemoprevention treatment plan.For example, if the caPCNA-derived peptides and the variants disclosedherein are not significantly toxic to normal cells, then higher dosesgenerally used for diagnosed cancer patients are used forchemoprevention in high-risk individuals. If the caPCNA-derived peptidesand the variants disclosed herein are somewhat toxic, then depending onthe toxicity, a relatively lower dosage as compared to a cancer therapydose is used for the chemopreventative treatment. In addition, thedosage may vary for example, depending on the cancer type, the stage atwhich the individual is being treated, mode of delivery, and any otherclinical data such as age, general health of the individual and thelike.

For example, caPCNA expression is readily observed in ChronicMyelogenous Leukemia (CML) patients, staged as being 3 years fromundergoing conversion to the acute phase of the disease. Thus, thecaPCNA peptides and the variants are also capable of reducing theviability of these types of early transformed cells, and prior todevelopment of a clinically recognized or detectable cancer.

Generally, the caPCNA-derived peptides or the variants are administeredby a suitable method to eliminate or reduce the number of earlytransformed cells that are likely to express the caPCNA isoform andlikely to become cancerous. This type of preventive administration isuseful in the clinical management of individuals belonging to “highrisk” groups such as those with familial types of cancer including thoseof epithelial (e.g., various adenocarcinomas) or mesothelial origin(e.g., various sarcomas), and/or individuals exposed to variousenvironmental toxicants derived from either their work or home orregional environments, and/or individuals treated for a cancer and whoare being monitored for recurrence of the disease (i.e., breaking out ofremission). Because only cancer cells or cells undergoing thetransformation process express the caPCNA isoform, these transformedcells that are sensitive to the caPCNA-derived peptides and variants areselectively targeted and killed, while the viability of non-transformedcells is not likely to be substantially affected by the caPCNAderived-peptides and variants thereof.

Long term benchmarks for clinical benefits of using the caPCNA-derivedpeptides and variants as chemo-preventative agents would include forexample, decrease in the number of “high risk” patients developingparticular malignancies, reductions in the number of cancer recurrencesfollowing excision of primary cancer lesions, reductions in the numberof occurrences of tumors resulting from exposure to carcinogenic ormutagenic substances encountered within the home, work, or regionalenvironment for individuals and reductions in the number of occurrencesof secondary tumors resulting from chemotherapeutic administration ofagents to treat unrelated cancers.

For example, co-expression of both p16^(INK4) (Bean et al., (2007),Clin. Cancer Res.; 13(22), 6834-41) and caPCNA in what appears to benon-cancer cells serves a marker for identification pre-cancerous orpre-malignant cells for chemoprevention. For example, the expression ofthe protein p16^(INK4) indicates that expression of this proteinprecedes transformation of the cell expressing p16^(INK4) to a cancerphenotype. Individual molecular changes occurring early in thetransformation process may predispose these cells to becoming cancercells, and that identification of such a change in gene expression is anindicator (marker) of the pending morphologically recognizabletransformation event at an early stage. Thus, early molecularalterations associated with the transformation event, while having nodiscernable effect on the morphology of the cells beginning thetransformation process, nonetheless are geared towards undergoingtransformation into a cancer cell. Since expression of caPCNA isuniversally associated with the malignant transformation process (allcancer cells examined to date express the caPCNA isoform), andoccasionally one or more normal breast epithelial cells within a ductthat is adjacent (within 1 cm) to breast cancer epithelial cells willalso express caPCNA. Co-expression of p16^(INK4) in these caPCNAexpressing cells, and absence from the non-expressing cells, may serveas a marker for the transformation process. Such “normal” cellsexpressing caPCNA are suitable targets to use the compositions disclosedherein to selectively kill the caPCNA expressing “normal” cells beforethey are malignant.

Therefore, the caPCNA-derived peptides or the variants disclosed hereinpresent a viable chemopreventative option to reduce the overalloccurrence of cancer in tissue types that are likely to express caPCNAisoform at an early stage. A practitioner or a clinician can readilydetermine if the individual or a tissue biopsy expresses caPCNA by usinga caPCNA-specific detection method e.g., caPCNA-specific antibodiesdisclosed in international patent application publication WO2006/116631or based on caPCNA isoform post-translational modifications disclosed inInternational Patent Application Publication WO 2007/002574, thecontents of both the publications are herein incorporated by referencein their entirety.

Example 6 Cytotoxic Effects of Cell-Permeable caPCNA-Derived PeptideVariants on Breast Cancer Cells Harboring BRCA1 Mutations

caPeptides are considered to be effective in cancer cells harboringmutations in DNA repair proteins. Inhibiting DNA repair proteinsrepresents a viable target for anti-cancer therapy. For example,inhibitors of a DNA repair protein Poly (ADP-ribose) polymerase family,member I, (PARP1) are used as anti-cancer therapeutic agents. caPCNApeptides that selectively interact with caPCNA isoform in malignant orpre-malignant cells are useful either as a monotherapy or a combinationtherapy with one or more chemotherapy agents such as cisplatin.

Breast cancer cells from a patient harboring a hereditary mutation inBRCA1, a major component of DNA double strand break repair (HCC1937cells). HCC1937 is a near-tetraploid cell line from mammary gland whosecells are homozygous for a frameshift mutation in BRCA1. The cell lineis available at ATCC.

As a genetically matched control, wild-type BRCA1 has been transducedinto HCC1937 cells (referred to as HCC1937+wild-type BRCA1).HCC1937+wild-type BRCA1 cells were selected for maintenance of thevector using 1 μg/mL puromycin. All cells in these assays were used atpassage numbers <25.

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assays were conducted with caPeptides at a variety of time points bothalone and in combination with a DNA damaging agent e.g., cisplatin inHCC1937 and HCC1937+wild-type BRCA1 cells. The MTT assay is based on theability of a mitochondrial dehydrogenase enzyme from viable cells tocleave the tetrazolium rings of the pale yellow MTT dye and form darkblue formazan crystals. These formazan crystals are largely impermeableto cell membranes and thus accumulate within healthy cells.Solubilization of the cells by the addition of a detergent results inthe liberation and solubilization of the crystals. The number ofproliferating cells is directly proportional to the level of formazanproduct created. The color can then be quantified using a simplecolorimetric assay. The results are read on a multiwall scanningspectrophotometer (ELISA reader). The peptides used in these experimentsincluding caPeptide, Q131A (LGIPEAEY (SEQ ID NO: 5)), and P129A(LGIAEQEY (SEQ ID NO: 3)) were ordered and obtained from Anaspec(Fremont, Calif.). caPeptide was dissolved in sterile 1× PBS solution tocreate a 1.5 mM stock concentration. caPeptides with alaninesubstitutions (e.g., P129A (SEQ ID NO: 3) and Q131A (SEQ ID NO: 5)) weredissolved in sterile 1×PBS solution to create 1.0 mM stock solutions.

Cisplatin powder was dissolved in sterile 1×PBS to create a 3.3 mM stocksolution. The stock peptide and cisplatin solutions were sterilefiltered using a 0.2 μM filter, aliquoted, and stored at −20° C. untiluse in the MTT experiments.

MTT assays: The efficacy of caPeptides were tested using the MTT assays:HCC1937 and HCC137+wildtype BRCA1 cells were grown in DMEM 1×(supplemented with 10% fetal bovine serum and 5%penicillin/streptomycin) and were plated at a densities of 5.5×10³ or7×10³, respectively, in 96 well plates (100 μL total volume per well)and allowed to attach overnight in a 37° C. incubator (5% CO₂). Then,caPeptide stock solution was diluted to final working concentrations inDMEM 1× (10% fetal bovine serum and 5% penicillin/streptomycin) of 150μM, 100 μM, 75 μM, 50 μM, and 25 μM. Media was removed from cells andreplaced with media containing caPeptide at various concentrations (200μL total volume per well). Cells were cultured with caPeptide-containingmedia for 24, 48 or 72 hours. At the appropriate treatment timepoint, 20μL MTT (5 mg/mL in PBS) reagent was added to each well and plates wereincubated for an additional 4 hours. Media/dye solution was thenaspirated and 200 μL DMSO was added to each well. Plates were rocked atroom temperature for five minutes to dissolve crystals and transferredto an ELISA plate reader. Absorbance was measured at 550 nm. The extentto which caPeptides inhibited cell proliferation was calculated as apercentage of the absorbance in each well containing caPeptide relativeto wells containing no caPeptide (negative control) for both HCC1937 andHCC1937+wild-type BRCA1 cells.

The negative control wells were assigned a value of 100%. The data werederived from a total of 3 experiments done at 24 and 48 hour timepointsand two experiments done at the 72 hour timepoint. The data shown hereare representative of results obtained in each cell line at thesetimepoints.

Efficacy of P129A (SEQ ID NO: 3) and Q131A (SEQ ID NO: 5) caPCNApeptides: Cells were cultured and plated in 96 well plates as describedherein. P129A (SEQ ID NO: 3) or Q131A (SEQ ID NO: 5) stock solution wasdiluted to final working concentrations in DMEM 1× (10% fetal bovineserum and 5% penicillin/streptomycin) of 100 μM, 75 μM, 50 μM, 25 μM,and 12.5 μM. Media was removed from cells and replaced with mediacontaining P129A (SEQ ID NO: 3) or Q131A (SEQ ID NO: 5) caPCNA peptidesat various concentrations (200 μL total volume per well) and cells werecultured with peptide-containing media for 24 hours. After 24 hours, theMTT assay was completed and data analysis was performed as describedherein.

Results: HCC1937 cells show a trend of increased sensitivity tocaPeptides at 24, 48, and 72 hour treatment timepoints in comparison toHCC1937+wild-type BRCA1 cells (FIGS. 10A-B). caPeptides with alaninesubstitutions at particular amino acid residues (P129A (SEQ ID NO: 3)and Q131A (SEQ ID NO: 5)) show enhanced efficacy in comparison towild-type peptides, and Q131A appears to be more effective of thesepeptides tested.

CaPeptide treatment inhibits colony formation in both HCC1937 andHCC1937+wild-type BRCA1 cells. This effect appears to be more pronouncedin HCC1937 cells lacking the wildtype BRCA1. These data suggest thatpeptides derived against a cancer-associated epitope of PCNA haveanticancer uses, and are capable of being particularly effective intreatment of cancers harboring mutations in DNA repair proteins. Inaddition, these peptides are capable of acting synergistically (i.e.,synergistic inhibition of tumor cells) with DNA damagingchemotherapeutics, for example, like cisplatin and others to reduce theIC50 of these chemotherapeutic agents. caPCNA Peptides therefore reducetoxicity to patients without compromising treatment efficacy.

Example 7 Efficacy of caPeptides Used in Combination with Cisplatin onBreast Cancer Cells Harboring BRCA1 Mutations

The purpose of these experiments was to determine whether caPeptides ata fixed concentration could lower the IC50 of cisplatin in HCC1937 andHCC1937+wild-type BRCA1 cells. Cells were cultured and plated in 96 wellplates as described herein. Then, cisplatin stock solution was dilutedto final working concentrations in DMEM 1× (10% fetal bovine serum and5% penicillin/streptomycin) of 200 μM, 100 μM, 75 μM, 50 μM, 25 μM, and12.5 μM. caPeptide stock solution was diluted to final workingconcentrations in DMEM 1× (10% fetal bovine serum and 5%penicillin/streptomycin) of 60 μM or 25 uM. Media was removed from cellsand replaced with media containing cisplatin alone, cisplatin and 60 μMcaPeptide added simultaneously, or 25 μM caPeptide (pretreatment)followed by addition of cisplatin into the media at increasingconcentrations after 3 or 6 hours. Cells treated with cisplatin alone orcisplatin plus 60 μM caPeptide treatment were incubated with peptideand/or cisplatin containing media for both 24 and 48 hour timepoints.Cells treated with 25 μM caPeptide pretreatment plus cisplatin wereincubated with media containing caPeptide and cisplatin for 24 hours. Atthe appropriate treatment timepoint, the MTT assay was completed anddata analysis was performed as described herein. The data detailing theeffects of cisplatin on these cell lines were derived from multipleexperiments. The data showing the effects of cisplatin in combinationwith 60 μM of caPeptide and cisplatin and 25 μM caPeptide pretreatmentwas experimentally verified.

Clonogeizic survival assay: Clonogenic survival after caPeptidetreatment: The purpose of this experiment was to determine the effectsof high caPeptide treatment (100 μM caPeptide for 48 hours) on abilityof cells plated at low density to proliferate and form colonies. HCC1937and HCC1937+wild-type BRCA1 cells were plated in 6-well plates andallowed to attach overnight at 37° C.

Then, media was removed and replaced with 100 μM caPeptide in DMEM 1×(10% fetal bovine serum and 5% penicillin/streptomycin). Untreated cellswere used as a negative control. Plates were incubated with mediacontaining peptide for 48 hours. Live cells were then collected,counted, plated at low density (1×10 cells in 10 cm2 dishes) and allowedto grow in DMEM 1× (20% fetal bovine serum, 5% penicillin/streptomycin)for 13 days. After 13-day incubation, media was removed and plates werewashed twice with 1×PBS. Cells were fixed with 70% EtOH for 10 minutes,dried, and stained for 1 hour with 20% Giemsa diluted in MilliQ water.Dye was removed; plates were rinsed, dried overnight and photographed.Images shown are representative of results obtained for treated anduntreated HCC1937 and HCC1937+wild-type BRCA1 cells.

HCC1937 and HCC1937+wild-type BRCA1 cells are relatively resistant tothe effects of cisplatin (FIGS. 10A, 10B). 60 μM caPeptide addedsimultaneously with cisplatin treatment lowers the IC50 of cisplatin andthis effect was more pronounced in HCC1937 cells lacking wild-type BRCA1(FIGS. 11A-B). Administration of caPCNA peptides induces cell death incancerous cells and augments the beneficial effects ofchemotherapeutics. caPCNA peptides are particularly effective in cellsharboring mutations in DNA repair proteins, for example, BRCA1.

TABLE 1 Exemplary caPCNA peptide domainscontaining the amino acid 126-133 region.PCNA Sequence 111-125 (SEQ ID NO: 42)LVFEAPNQEK VSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRD LSHIGDAVVI SCAKDGVKFS ASGELGNGNI KLSQTSNVDK EEEAVTIEMN(SEQ ID NO: 43) PCNA Sequence 118-135 (SEQ ID NO: 44)LVFEAPNQEK VSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRD LSHIGDAVVI SCAKDGVKFS ASGELGNGNI KLSQTSNVDK EEEAVTIEMN(SEQ ID NO: 43) PCNA Sequence 121-133 (SEQ ID NO: 45)LVFEAPNQEK VSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRD LSHIGDAVVI SCAKDGVKFS ASGELGNGNI KLSQTSNVDK EEEAVTIEMN(SEQ ID NO: 43) PCNA Sequence 126-133 (SEQ ID NO: 1)LVFEAPNQEK VSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRD LSHIGDAVVI SCAKDGVKFS ASGELGNGNI KLSQTSNVDK EEEAVTIEMN(SEQ ID NO: 43) PCNA Sequence 126-143 (SEQ ID NO: 46)LVFEAPNQEK VSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRD LSHIGDAVVI SCAKDGVKFS ASGELGNGNI KLSQTSNVDK EEEAVTIEMN(SEQ ID NO: 43) PCNA Sequence 126-153 (SEQ ID NO: 47)LVFEAPNQEK VSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRD LSHIGDAVVI SCAKDGVKFS ASGELGNGNI KLSQTSNVDK EEEAVTIEMN(SEQ ID NO: 43) PCNA Sequence 126-163 (SEQ ID NO: 48)LVFEAPNQEK VSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRD LSHIGDAVVI SCAKDGVKFS ASGELGNGNI KLSQTSNVDK EEEAVTIEMN(SEQ ID NO: 43)The regions containing the 126-133 sequence are shown as underlined.

TABLE 2 Amino acid sequences of R9-lined caPCNA-derived peptides. NameSequence R9 Alone RRRRRRRRR (SEQ ID NO: 13) caPeptide LGIPEQEY(SEQ ID NO: 1) R9-caPep RRRRRRRRRCCLGIPEQEY (SEQ ID NO: 49) R9-L126ARRRRRRRRRCCAGIPEQEY (SEQ ID NO: 50) R9-L127A RRRRRRRRRCCLAIPEQEY(SEQ ID NO: 51) R9-L128A RRRRRRRRRCCLGAPEQEY (SEQ ID NO: 52) R9-L129ARRRRRRRRRCCLGIAEQEY (SEQ ID NO: 53) R9-L130A RRRRRRRRRCCLGIPAQEY(SEQ ID NO: 54) R9-L131A RRRRRRRRRCCLGIPEAEY (SEQ ID NO: 55) R9-L132ARRRRRRRRRCCLGIPEQAY (SEQ ID NO: 56) R9-L133A RRRRRRRRRCCLGIPEQEA(SEQ ID NO: 57) FITC-R9- FITC-RRRRRRRRRCCLGIPEQ (SEQ ID NO: 49) caPep EY

TABLE 3 Cytotoxic effects of R9-linked caPCNA peptides. Arginine-linkedcaPCNA peptide and the % cancer Peptide designation respective alaninesubstitutions cell death 126-133 peptide 126- L G I P E Q E Y −133Unsub. Peptide R9- 1 2 3 4 5 6 7 8 43 ± 10 (SEQ ID NO: 58) A1-sub.Peptide R9- A 2 3 4 5 6 7 8 8 ± 9 (SEQ ID NO: 59) A2-sub. Peptide R9- 1A 3 4 5 6 7 8 47 ± 13 (SEQ ID NO: 60) A3-sub. Peptide R9- 1 2 A 4 5 6 78 21 ± 12 (SEQ ID NO: 61) A4-sub. Peptide R9- 1 2 3 A 5 6 7 8 80 ± 7 (SEQ ID NO: 62) A5-sub. Peptide R9- 1 2 3 4 A 6 7 8 45 ± 14 (SEQ ID NO:63) A6-sub. Peptide R9- 1 2 3 4 5 A 7 8 93 ± 7  (SEQ ID NO: 64) A7-sub.Peptide R9- 1 2 3 4 5 6 A 8 88 ± 4  (SEQ ID NO: 65) A8-sub. Peptide R9-1 2 3 4 5 6 7 A 7 ± 6 (SEQ ID NO: 66) Negative control Scrambled peptide5 ± 4 R9 refers to a cell penetrating peptide that includes apolyarginine sequence e.g., nine contiguous arginine residues. Alaninesubstitutions in the caPCNA peptide affect its cytotoxic action. U937cells were treated with each alanine substituted peptide and thescrambled peptide and % cell death evaluated by flow cytometry. Thescrambled peptide and alanine substitutions at positions aa126 or aa133show reduced cytotoxic activity. Substitutions at aa129, aa131, or aa132cause an increase in cytotoxicity of the peptide. Substitutions thatresult in a neutral change include aa127 and aa130. Substitution ataa128 results in a decrease in cytotoxic action but not to the levels ofaa126 or aa133.

TABLE 4 Cell permeable or cell-penetrating peptides References (eachis incorporated by reference in its Name Peptide sequence entirety) R9RRRRRRRRR (SEQ ID NO: 13) Penetratin™ RQIKIWFQNRRMKWKK U.S. Pat. No.(SEQ ID NO: 16) 5,888,762 Tat GRKKRRQRRRPPQ U.S. Pat. Nos.(SEQ ID NO: 17) 5,804,604 and 5,674,980 TAT (47-57) YGRKKRRQRRRWender, PA. et (SEQ ID NO: 67) al. Proc. Natl. Acad. Sci. USA97, 13003 (2000) Tat (48-57) GRKKRRQRRR Hottiger, M. and (SEQ ID NO: 19)G. Nabel, J. Virol. 72, 8252 (1998). Tat (Npys)YGRKKRRQRRRGGG-C(Npys)-NH2 (SEQ ID NO: 68) Transportan™GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18) VP22DAATATRGRSAASRPTERPRAPARSASRPRRPVD WO 97/05265 (SEQ ID NO: 69) MAPKLALKLALKALKAALKLA (SEQ ID NO: 70) KALA WEAKLAKALAKALAKHLAKALAKALKACEA(SEQ ID NO: 71) ppTG20 GLFRALLRLLRSLWRLLLRA (SEQ ID NO: 72) TrimerVRLPPP (SEQ ID NO: 73) P1 MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 74)MPG GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 75) Pep-1KETWWETWWTEWSQPKKKRKV Fischer, R. et al. (SEQ ID NO: 76) Chem. Bio.Chem. 6, 2126 (2005). hCT LGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO: 77) C105YCSIPPEVKFNKPFVYLI Rhee and Davis, (SEQ ID NO: 78) (2006) J. Biol.Chem. 281, 1233 105Y SIPPEVKFNKPFVYLI Boland, K. et al. (SEQ ID NO: 79)J. Biol. Chem. 270, 28022 (1995) Lipid KKAAAVLLPVLLAAP and its D-isomerMembrane (SEQ ID NO: 80) Translocating Peptide Nuclear PKKKRKVlocalization (SEQ ID NO: 81) RVG YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGKumar, P. et al. (SEQ ID NO: 82) Nature 448, 39 (2007). TransdermalACSSSPSKHCG Chen, Y. et al. Peptide (SEQ ID NO: 83) Nat. Biotechnol.24, 455 (2006). Antennapedia KKWKMRRNQFWVKVQRG Kanovsky, M. et Leader(SEQ ID NO: 84) al. Proc. Natl. Peptide (CT) Acad. Sci. 98,12438 (2001). Antennapedia  RQIKIWFQNRRMKWKK Jain, M. et al. Peptide(SEQ ID NO: 85) Cancer Res. 65, 7840 (2005). SynB1 RGGRLSYSRRRFSTSTGRA(SEQ ID NO: 86) The peptides listed above can be synthesized or arecommercially available (e.g., AnaSpec, San Jose, CA) with an NH₂ moietyfor coupling with the peptide variants disclosed herein.

TABLE 5 List of exemplary amino acid conserved substitutions Amino AcidCode Conserved Substitutions Alanine A D-Ala, Gly, beta-Ala, L-Cys,D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile,D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu,Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-GlnCysteine C D-Cys, S—Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln,Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp,Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, AcpIsoleucine I D-Ille, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu,Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S—Me-Cys, Ile,D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa,His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4 or5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylic acid Serine S De-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Lle,D-Lle, Met, D-Met

TABLE 6 Preferential killing of cancer cells by R9-caPeptide Cell line %Dead Cells MCF 10A (non- 18.2 malignant) MCF 7 (breast cancer) 91.5

1. A therapeutic composition for reducing cellular proliferation ofmalignant cells, the composition comprising a peptide molecule fused orlinked to a cell penetrating peptide, wherein the peptide moleculecomprises an amino acid sequence selected from the group consisting ofLGIPEQEY (SEQ ID NO: 1), LAIPEQEY (SEQ ID NO: 2), LGIAEQEY (SEQ ID NO:3), LGIPAQEY (SEQ ID NO: 4), LGIPEAEY (SEQ ID NO: 5), LGIPEQAY (SEQ IDNO: 6), LGIAEAEY (SEQ ID NO: 7), LGIPEAAY (SEQ ID NO: 8), LGIAEQAY (SEQID NO: 9), and LGIAEAAY (SEQ ID NO: 10), and wherein the cellpenetrating peptide comprises an amino acid sequence selected from thegroup consisting of RRRRRRR (SEQ ID NO: 11), RRRRRRRR (SEQ ID NO: 12),RRRRRRRRR (SEQ ID NO: 13), RRRRRRRRRR (SEQ ID NO: 14), RRRRRRRRRRR (SEQID NO: 15), RQIKIWFQNRRMKWKK (SEQ ID NO: 16), GRKKRRQRRRPPQ (SEQ ID NO:17), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18), and GRKKRRQRRR (SEQ IDNO: 19).
 2. The composition of claim 1, wherein the amino acid sequenceof the cell penetrating peptide comprises one or more amino acidD-isomers.
 3. The composition of claim 1, wherein the cell penetratingpeptide is covalently linked or conjugated to the peptide molecule. 4.The composition of claim 1, wherein the cell penetrating peptide isrecombinantly fused with the peptide molecule.
 5. The composition ofclaim 1, wherein the peptide molecule further comprises a cell surfacetargeting factor.
 6. The composition of claim 1, further comprising achemotherapeutic agent.
 7. The composition of claim 6, wherein thechemotherapeutic agent is a DNA damaging agent.
 8. The composition ofclaim 7, wherein the DNA damaging agent is selected from the groupconsisting of doxorubicin, irinotecan, cyclophosphamide, chlorambucil,melphalan, methotrexate, cytarabine, fludarabine, 6-mercaptopurine,5-fluorouracil, capecytabine, cisplatin, carboplatin, oxaliplatin, and acombination thereof.
 9. A method In vitro of reducing cellularproliferation or inducing cell death of a cancer cell or a pre-malignantcell expressing cancer-specific proliferating cell nuclear antigen(caPCNA), the method comprising administering a therapeuticallyeffective amount of a composition comprising a peptide molecule fused orlinked to a cell penetrating peptide, wherein the peptide moleculecomprises an amino acid sequence selected from LGIPEQEY (SEQ ID NO: 1),LAIPEQEY (SEQ ID NO: 2), LGIAEQEY (SEQ ID NO: 3), LGIPAQEY (SEQ ID NO:4), LGIPEAEY (SEQ ID NO: 5), LGIPEQAY (SEQ ID NO: 6), LGIAEAEY (SEQ IDNO: 7), LGIPEAAY (SEQ ID NO: 8), LGIAEQAY (SEQ ID NO: 9), and LGIAEAAY(SEQ ID NO: 10), and wherein the cell penetrating peptide comprises anamino acid sequence selected from the group consisting of RRRRRRR (SEQID NO: 11), RRRRRRRR (SEQ ID NO: 12), RRRRRRRRR (SEQ ID NO: 13),RRRRRRRRRR (SEQ ID NO: 14), RRRRRRRRRRR (SEQ ID NO: 15),RQIKIWFQNRRMKWKK (SEQ ID NO: 16), GRKKRRQRRRPPQ (SEQ ID NO: 17),GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18), and GRKKRRQRRR (SEQ ID NO:19), to contact the cancer or pre-malignant cell expressing caPCNA. 10.The method of claim 9, wherein the cancer or premalignant cell has amutation in a DNA repair protein.
 11. The method of claim 10, whereinthe DNA repair protein participates in homologous recombination.
 12. Themethod of claim 11, wherein the protein is BRCA1.
 13. The method ofclaim 9, further comprising administering a chemotherapeutic agent. 14.The method of claim 13, wherein the chemotherapeutic agent is a DNAdamaging agent.
 15. The method of claim 14, wherein the DNA damagingagent is selected from the group consisting of doxorubicin, irinotecan,cyclophosphamide, chlorambucil, melphalan, methotrexate, cytarabine,fludarabine, 6-mercaptopurine, 5-fluorouracil, capecytabine, cisplatin,carboplatin, oxaliplatin, and combinations thereof.
 16. The method ofclaim 9, wherein the cancer or pre-malignant cell is a breast, colon,lung, ovary, prostate, or blood cell.